A method and system for cryopreservation to achieve uniform viability and biological activity

ABSTRACT

A method and system for controlled rate freezing and nucleation of biological materials is provided. The presently disclosed system and method provides the ability to rapidly cool the materials contained in vials or other containers within a cooling unit via forced convective cooling and optionally simultaneous pressure drop using uniform and unidirectional flow of cryogen in proximity to the plurality of vials disposed within a cooling unit. The rapid cooling of the biological materials is achieved by precisely controlling and adjusting the temperature of the cryogen being introduced to the system as well as the chamber pressure as a function of time.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation-in-part application of U.S.patent application Ser. No. 12/266,760 filed Nov. 7, 2008 and alsoclaims priority from U.S. provisional patent application Ser. No.61/480,647 filed Apr. 29, 2011 the disclosure of both applications arealso fully incorporated by reference herein.

FIELD OF THE INVENTION

The present invention broadly relates to a cryopreservation process, andmore particularly, to a method and system for providing controlled ratefreezing and nucleation control of biological materials to minimize celldamage resulting from intercellular ice formation and solute effectsthat arise during the cryopreservation process.

BACKGROUND

Cryopreservation is a process used to stabilize biological materials atvery low temperatures. Previous attempts to freeze biological materials,such as living cells often results in a significant loss of cellviability and in some cases as much as 80% or more loss of cell activityand viability.

Cell damage during cryopreservation usually occurs as a result ofintracellular ice formation within the living cell during the freezingstep or during subsequent recrystallization. Rapid cooling often leadsto formation of more intracellular ice since water molecules are notfully migrated out of the cell during the short timeframe associatedwith the rapid cool-down rates. Intercellular ice formation also canarise during recrystallization that occurs during the warming or thawingcycles. If too much water remains inside the living cell, damage due toinitial ice crystal formation during the rapid cooling phase andsubsequent recrystallization during warming phases can occur and suchdamage is usually lethal.

On the other hand, slow cooling profiles during cryopreservation oftenresults in an increase in the solute effects where excess water ismigrated out of the cells. Excess water migrating out of the cellsadversely affects the cells due to an increase in osmotic imbalance.Thus, cell damage occurs as a result of osmotic imbalances which can bedetrimental to cell survival and ultimately lead to cell damage and aloss of cell viability.

Current cryopreservation techniques involve using either conductivebased cryogenic cooling equipment such as a cold shelf or lyophilizertype freezer unit or convective based cryogenic cooling equipment suchas controlled rate freezers and cryo-cooler units. Such equipment,however, is only suitable for relatively small volume capacities and isnot suitable for commercial scale production and preservation ofbiological materials such as therapeutic cell lines. For example, thelargest commercially available controlled rate freezer suitable for usewith biological materials holds only about 8000 or so closely packedvials. One such system is the Kryo 1060-380 capable of storing 8000×2 mlampoules. Such existing controlled rate freezers, including the Kryo1060 series, also suffer from the non-uniformity in cooling vial to vialdue, in part, to the non-uniform flow of cryogen within the freezers andthe requirement for close packing of the vials within the freezer. Thesize of individual conventional freezers is limited due to thesenon-uniform effects. As conventional controlled rate freezers are scaledup in size, the non-uniformities in cooling increase. Consequently, thesize of conventional controlled rate freezers must be limited to preventnon-uniform sample-to-sample properties due to non-uniform cooling ofeach sample. The only effective way to further increase the quantity ofsamples processed at once using conventional controlled rate freezers isto use multiple controlled rate freezers.

Many conventional freezing systems utilize internal fans to dispersecryogen around the unit and deliver the refrigeration to the vials viaconvection. Such convection based cooling or freezing systems cannotachieve temperature uniformity as the vials are often located at variousdistances from the internal fan or packed in the shadow of other vialsor trays. Vials of biological material exposed to high velocityturbulent flow of cryogen are typically cooled at a different rate andoften much faster than vials situated further away from the fan.

There are also existing lyophilizer type of control rate freezers thatcan handle large volumes of vials but typically rely on thermalconduction between cold shelves in the lyophilizer unit and the vials.However, it is impossible to provide a uniform conductive surface areaon the bottom of each glass vial since most glass vial bottoms areconcave. Therefore, temperature variations during the freezing processfrom vial to vial are the biggest drawback for these types of equipment.Furthermore, the cooling rate can be painfully slow due to the verysmall conductive surface of the vial that remains in contact with thecold shelves.

Prior attempts to mitigate the loss of cell activity and viabilityinvolved the use of cryoprotective additives such as DSMO and glycerol.Use of such cryoprotectives during the cryopreservation process hasdemonstrated a reduction in cell losses attributable to more suitablefreezing and subsequent thawing cycles. However, many cryoprotectantssuch as DSMO are toxic to human cells and are otherwise not suitable foruse in whole cell therapies. Disadvantageously, cryoprotectants also adda degree of complexity and associated cost to the cell production andpreservation process. Also, cryoprotectants alone, have not eradicatedthe problem of loss of cell activity and viability.

Another problem associated with the above mentioned systems is a lack ofcontrol with respect to the uniformity of the nucleation temperaturebetween the multiple vials. This variability in the nucleationtemperature of the multiple vials can lead to non-uniform vial-to-vialproperties. Such properties can include cell activity and viability aswell as the crystal structure of the frozen material and the time neededto complete a freeze drying process. Consequently, controlling thegenerally random process of nucleation in the freezing stage of acryopreservation, lyophilization, or freeze-drying process to increasethe product uniformity from vial-to-vial in the finished product wouldbe highly desirable in the art.

In a typical pharmaceutical freeze-drying process, multiple vialscontaining a common aqueous solution are placed on shelves that arecooled, generally at a controlled rate, to low temperatures. The aqueoussolution in each vial is cooled below the thermodynamic freezingtemperature of the solution and remains in a sub-cooled metastableliquid state until nucleation occurs.

The range of nucleation temperatures across the vials is distributedrandomly between a temperature near the thermodynamic freezingtemperature and some value significantly (e.g., up to about 30° C.)lower than the thermodynamic freezing temperature. This distribution ofnucleation temperatures causes vial-to-vial variation in ice crystalstructure and ultimately the physical properties of the lyophilizedproduct. Furthermore, the drying stage of the freeze-drying process mustbe excessively long to accommodate the range of ice crystal sizes andstructures produced by the natural stochastic nucleation phenomenon.

Additives have been used to increase the nucleation temperature ofsub-cooled solutions. These additives can take many forms. It is wellknown that certain bacteria (e.g., Pseudomonas syringae) synthesizeproteins that help nucleate ice formation in sub-cooled aqueoussolutions. Either the bacteria or their isolated proteins can be addedto solutions to increase the nucleation temperature. Several inorganicadditives also demonstrate a nucleating effect; the most common suchadditive is silver iodide, AgI. In general, any additive or contaminanthas the potential to serve as a nucleating agent. For instance,lyophilization vials prepared in environments containing highparticulate levels will generally nucleate and freeze at a lower degreeof sub-cooling than vials prepared in low particulate environments.

All the nucleating agents described above are known as or labeled“additives,” because they change the composition of the medium in whichthey nucleate a phase transition. These additives are not typicallyacceptable or desirable for FDA regulated and approved freeze-driedpharmaceutical products. These additives also do not provide controlover the time and temperature during which the vials nucleate andfreeze. Rather, the additives operate primarily to increase the averagenucleation temperature of the vials (e.g. as freezing temperaturedepressants).

Ice crystals can themselves act as nucleating agents for ice formationin sub-cooled aqueous solutions. In the “ice fog” method, a humidfreeze-dryer is filled with a cold gas to produce a vapor suspension ofsmall ice particles. The ice particles are transported into the vialsand initiate nucleation when they contact the fluid interface.

The “ice fog” method does not control the nucleation of multiple vialssimultaneously at a controlled time and temperature. In other words, thenucleation event does not occur concurrently or instantaneously withinall vials upon introduction of the cold vapor into the freeze-dryer. Theice crystals will take some time to work their way into each of thevials to initiate nucleation, and transport times are likely to bedifferent for vials in different locations within the freeze-dryer. Forlarge scale industrial freeze-dryers, implementation of the “ice fog”method would require system design changes as internal convectiondevices may be required to assist in a more uniform distribution of the“ice fog” throughout the freeze-dryer. When the freeze-dryer shelves arecontinually cooled, the time difference between when the first vialfreezes and the last vial freezes creates a difference in thetemperature between vials, which will also increase the vial-to-vialnon-uniformity in the final freeze-dried products.

Vial pre-treatment by scoring, scratching, or roughening has also beenused to lower the degree of sub-cooling required for nucleation. As withthe other prior art methods, vial pre-treatment also does not impart anydegree of control over the time and temperature when the individualvials nucleate and freeze, but instead only increases the averagenucleation temperature of all vials.

Vibration has also been used to nucleate a phase transition in ametastable material. Vibration sufficient to induce nucleation occurs atfrequencies above 10 kHz and can be produced using a variety ofequipment. Often vibrations in this frequency range are termed“ultrasonic,” although frequencies in the range 10 kHz to 20 kHz aretypically within the audible range of humans. Ultrasonic vibration oftenproduces cavitation, or the formation of small gas bubbles, in asub-cooled solution. In the transient or inertial cavitation regime, thegas bubbles rapidly grow and collapse, causing very high localizedpressure and temperature fluctuations. The ability of ultrasonicvibration to induce nucleation in a metastable material is oftenattributed to the disturbances caused by transient cavitation. The othercavitation regime, termed stable or non-inertial, is characterized bybubbles that exhibit stable volume or shape oscillations withoutcollapse. U.S. Patent Application 20020031577 A1 discloses thatultrasonic vibration can induce nucleation even in the stable cavitationregime, but no explanation of the phenomenon is offered. GB PatentApplication 2400901A also discloses that the likelihood of causingcavitation, and hence nucleation, in a solution using vibrations withfrequencies above 10 kHz may be increased by reducing the ambientpressure around the solution or dissolving a volatile fluid in thesolution.

An electrofreezing method has also been used in the past to inducenucleation in sub-cooled liquids. Electrofreezing is generallyaccomplished by delivering relatively high electric fields (1 V/nm) in acontinuous or pulsed manner between narrowly spaced electrodes immersedin a sub-cooled liquid or solution. Drawbacks associated with anelectrofreezing process in typical lyophilization applications includethe relative complexity and cost to implement and maintain, particularlyfor lyophilization applications using multiple vials or containers.Also, electrofreezing cannot be directly applied to solutions containingionic species (e.g., NaCl).

Recently, there have been studies that examined the concept of‘vacuum-induced surface freezing’ (See e.g., U.S. Pat. No. 6,684,524).In such ‘vacuum induced surface freezing’, vials containing an aqueoussolution are loaded on a temperature controlled shelf in a freeze-dryerand held initially at about 10 degrees Celsius. The freeze-dryingchamber is then evacuated to near vacuum pressure (e.g., 1 mbar) whichcauses surface freezing of the aqueous solutions to depths of a fewmillimeters. Subsequent release of vacuum and decrease of shelftemperature below the solution freezing point allows growth of icecrystals from the pre-frozen surface layer through the remainder of thesolution. A major drawback for implementing this ‘vacuum induced surfacefreezing’ process in a typical lyophilization application is the highrisk of violently boiling or out-gassing the solution under statedconditions.

Improved control of the nucleation process could enable the freezing ofall unfrozen solution containers in a cryogenic chiller or freeze-dryerto occur within a narrower temperature and time range, thereby yieldinga product with greater uniformity from sample-to-sample. With regard tofreeze-drying systems, controlling the minimum nucleation temperatureaffects the ice crystal structure formed within the vial and allows fora greatly accelerated freeze-drying process.

In view of the above, what is needed is a method and system to controlthe uniformity of the temperature profiles and nucleation temperaturesof the multiple containers so as to provide a more uniform finishedproduct sample-to-sample. Moreover, the system and method should be bothefficient and readily scalable to handle commercial scale production.

SUMMARY OF THE INVENTION

The invention may be characterized as a method of controlling a chillingor freezing process of biological material disposed in a plurality ofcontainers, comprising the steps of: (i) placing said plurality ofcontainers of said biological materials in a cooling area defined as anarea between parallel porous surfaces within a cooling chamber; (ii)mixing a liquid cryogen with a warmer gas to produce a cold cryogenicgas at a selected temperature profile, said temperature profilecorresponding to a desired cooling rate of said biological materialswithin said containers; (iii) delivering a unidirectional flow of saidcold cryogenic gas through one of the porous surfaces to said coolingarea between said parallel porous surfaces and generally parallel toeach of said plurality of containers to uniformly cool said biologicalmaterials within said containers; and (iv) promptly exhausting said gasfrom said cooling chamber via another parallel porous surface so as toprevent recirculation of said gas within said cooling area; whereingreater uniformity of said biological materials in each of saidplurality of containers is achieved.

The inventors have recognized and appreciated a need for deliveringuniform viability and/or desired biological activity to a plurality ofcontainers each containing a biological material during acryopreservation process on a commercial scale. Furthermore, theinventors have recognized and appreciated that it is possible to providea uniform enhanced viability and/or biological activity to thebiological material in each of the containers by uniformly controllingthe temperature profile and nucleation of freezing for each container.More generally, the inventors have recognized the advantages of a methodcapable of providing large scale commercial volumes of frozen biologicalmaterial exhibiting uniform enhanced viabilities and/or biologicalactivities. For purposes of this application, biological activity isdefined as the therapeutic, biologic, or biochemical effect of amaterial or constituents of the material on living matter. Such a methodis capable of being used for any number of different applications inaddition to cryopreservation.

In one exemplary embodiment, the uniformity of the viability and/orbiological activity in each container is maintained within ±5% duringthe freezing process regardless of the location in the cooling chamberwhere the material is frozen. The above example of uniformity ofviability and/or biological activity should not be construed as limitingwith regard to the current disclosure.

In another exemplary embodiment, a method of controlling a freezingprocess of biological material in a plurality of containers includesproviding a plurality of containers each holding a biological materialin a cooling chamber within a single system. The plurality of containersmay be at least 10,000, and often as many as 20,000, 50,000, or 100,000.The biological material in each of the plurality of containers is frozenwhile the containers are in the cooling chamber. The freezing process issuch that there is a uniform viability and/or biological activity of thefrozen biological material in each of the plurality of containers.

In a further exemplary embodiment, the uniform viability and/orbiological activity of the frozen biological material in each of theplurality of containers provides a uniformly enhanced viability and/orbiological activity.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein.

The foregoing and other aspects, embodiments, and features of thepresent teachings can be more fully understood from the followingdescription in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a schematic illustration of an embodiment of a uniform flowcryogenic chiller unit;

FIG. 2 is a detailed view of a cut-away portion of the uniform flowcryogenic chiller unit of FIG. 1 depicting the uniform flowcharacteristics of the cryogen gas proximate the vials of biologicalmaterials;

FIG. 3 is a diagram of an embodiment of a single batch uniform flowcryogenic chiller unit;

FIG. 4 is a schematic view of an embodiment of a multi-batch or largecommercial scale uniform flow cooling chamber;

FIG. 5 is a schematic view of another embodiment of a continuous typeuniform flow cooling unit;

FIGS. 6 through 8 depict selected temperature profiles of the cryogeniccold gas and corresponding relationship to the cooling rates ofbiological materials contained in multiple vials;

FIG. 9 depicts an embodiment of a multi-batch or commercial scaleuniform flow cooling system with more detailed views of the process andinstrumentation;

FIG. 10 depicts another embodiment of a multi-batch or commercial scaleuniform flow cooling system with more detailed views of the process andinstrumentation;

FIG. 11 is an illustrative temperature profile of the cryogenic cold gasduring a temperature quench induced nucleation freezing process;

FIG. 12 is a graph depicting the temperature profiles of differentsamples during a temperature quench induced nucleation freezing process;

FIG. 13 is a graph depicting the temperature profiles of differentsamples during a freezing process with no nucleation control;

FIG. 14 is a graph depicting the temperature versus time plot of asolution undergoing a stochastic nucleation process and further showingthe range of nucleation temperatures of the solution;

FIG. 15 is a graph depicting the temperature versus time plot of asolution undergoing an equilibrated cooling process with depressurizednucleation;

FIG. 16 is a graph depicting the temperature versus time plot of asolution undergoing a dynamic cooling process with depressurizednucleation;

FIG. 17 is a schematic representation of a lyophilization system inaccordance with the present invention;

FIG. 18 a depicts illustrative temperature and pressure profiles versustime of the cryogenic cold gas during a depressurization inducednucleation freezing process;

FIG. 18 b is a graph depicting the pressure profile and the temperatureprofiles of different samples during a depressurization inducednucleation freezing process;

FIG. 19 is a graph depicting cell viability versus pre-nucleationtemperature;

FIG. 20 is a graph depicting cell viability versus cold spiketemperature;

FIG. 21 is a graph depicting temperature profiles of samples subjectedto the different cold spike temperatures of FIG. 20;

FIG. 22 is a graph depicting cell viability versus cold spiketemperature and hold time;

FIG. 23 is a graph depicting temperature profiles of samples subjectedto the different cold spike conditions of FIG. 22;

FIG. 24 is a graph depicting cell viability versus post-nucleationholding temperature;

FIG. 25 is a graph depicting temperature profiles of samples subjectedto the different post-nucleation holding temperatures of FIG. 24;

FIG. 26 is a graph depicting cell viability versus post-nucleationholding time;

FIG. 27 is a graph depicting temperature profiles of samples subjectedto the different post-nucleation holding times of FIG. 26;

FIG. 28 is a graph depicting cell viability versus post-nucleationcooling rate following a 10 minute hold at −35° C.;

FIG. 29 is a graph depicting temperature profiles of samples subjectedto the different post-nucleation cooling rates of FIG. 28 after holdingat −35° C.;

FIG. 30 is a graph depicting cell viability versus post-nucleationcooling rate following a 10 minute hold at −10° C.;

FIG. 31 is a graph depicting temperature profiles of samples subjectedto the different post-nucleation cooling rates of FIG. 30 after holdingat −10° C.; and

FIG. 32 is a chart comparing cell recovery following freezing in thecontrolled rate freezing system in accordance of the present invention.

DETAILED DESCRIPTION

It should be understood that aspects of the invention are describedherein with reference to the figures, which show illustrativeembodiments in accordance with aspects of the invention. Theillustrative embodiments described herein are not necessarily intendedto show all aspects of the invention, but rather are used to describe afew illustrative embodiments. Thus, aspects of the invention are notintended to be construed narrowly in view of the illustrativeembodiments. It should be appreciated, then, that the various conceptsand embodiments introduced above and those discussed in greater detailbelow may be implemented in any of numerous ways, as the disclosedconcepts and embodiments are not limited to any particular manner ofimplementation. In addition, it should be understood that aspects of theinvention may be used alone or in any suitable combination with otheraspects of the invention.

Controlled Rate Freezing

Cryopreservation of biological materials typically involves rapidcooling of biological specimens from temperatures of 40° C. or more totemperatures of about −100° C. or lower. The specified temperatures,cool-down rates, and cooling profiles, expressed as temperature of thematerials as a function of time, are highly dependent on the specificbiological materials to be frozen. In most cryopreservation ofbiological materials, the freezing process must be precisely controlled.Uniformity in temperatures, cool-down rates, and cooling profiles fromcontainer to container and batch to batch is of utmost importance in theproduction process.

The presently disclosed method and system represents an improvement tocurrent cryopreservation processes for biological materials. Thepresently disclosed system and method provides the ability to rapidlycool the biological materials contained in vials or other containerswithin a cooling unit primarily via forced convective coolingsimultaneously using a uniform flow of cryogen in proximity to each ofthe plurality of vials disposed within the cooling unit. In addition,the present system and methods are capable of providing the rapidcooling of the biological materials over a wide range of cooling rateswhile simultaneously holding the temperature of the biological materialsat the prescribed and specified temperature.

More specifically, the rapid cooling of the biological materials isachieved by precisely controlling and adjusting the temperature of thecryogen being introduced to the system as a function of time. In onemode, the disclosed embodiments of the system are adapted to provide astepwise (quick) drop in cryogen temperature 102 (See FIG. 6) togenerate a higher degree of sub-cooling within the sample materials 100thereby minimizing the exothermic effects of the phase transition (e.g.water-to-ice transformation) in the vials. In another mode, thedisclosed embodiments of the present controlled rate freezing orcryogenic chilling system and method are adapted to provide a ramp downof cryogen cold gas temperature at a rate of about −4.5° C. per minute112 (See FIG. 7) and of about −5.0° C. per minute (See FIG. 8),respectfully in order to provide rapid cooling of the sample biologicalmaterials 110, 120 yet minimize any vial to vial variations intemperature.

Temperatures of the cold cryogen gas introduced to the cooling chamberor unit are adjusted or otherwise controlled by mixing a source ofliquid nitrogen with a source of warmer nitrogen gas just prior tointroduction of the cold cryogen gas to the cooling unit. The mixed flowis then introduced and dispersed throughout the cooling unit by means ofsuitable cryogen intake circuits, as described herein. The warmernitrogen gas is preferably either room temperature nitrogen gas from asupply source or nitrogen gas exiting from the cooling unit and recycledto the cryogen intake circuit. The warmer nitrogen gas mixed with thecold nitrogen liquid or gas also acts as a motive gas and preferably hasa volumetric flow rate many times that of the liquid or cold nitrogen.Through the appropriate mixing of the warmer nitrogen gas with thecooler nitrogen flow, the present system creates a uniform flow of thecryogen across the entire cooling area targeted by the cold cryogen gas.By recycling the nitrogen gas exiting the cooling unit(s), the presentlydisclosed system and method also offers a higher utilization efficiencyof the cryogen (e.g. nitrogen) than existing controlled rate freezers.

Given the uniform flow of the cold cryogen gas across all samples orvials of the biological material, it has been found that precise controlof the cold cryogen gas temperature and cryogen temperature gradient hasa direct correlation to the observed cooling rates of the biologicalmaterial within the cooling unit, for a given biological material. Forexample, when the cold cryogen gas temperature provided to the presentcooling unit is varied or ramped at about −4.5° C./min to about −5.0°C./min, an average cooling rate of the biological material ofapproximately −2.5° C./min is achieved with minimum vial-to-vialtemperature variations. (See FIGS. 7 and 8).

Turning now to FIGS. 1 and 2, there are depicted selected views of acooling unit, referred to as a uniform flow cryogenic chiller 10. Asseen therein, the uniform flow cryogenic chiller 10 includes a cryogenintake circuit 12 or conduit coupled to a source of cryogen (not shown).The uniform flow cryogenic chiller 10 further includes a base gasinjection box 14, a porous metal plate 16 disposed or set in or near thetop surface 17 of the gas injection box 14, and a corresponding gasremoval box 18 positioned immediately above the base gas injection box14 and porous metal plate 19 disposed therein. Alternatively, variousarrangements of supported polymeric membranes suitable to withstand thecryogenic temperatures or other perforated plates with mechanicallypunctured or chemically etched holes can be used in lieu of the porousmetal plates. Alternatively, the gas removal box 18 may include othercollection surfaces in lieu of the porous metal plate, such as a mesh,screen or open surface or area leading to the exhaust manifold 34.

The porous metal plate 16 associated with the gas injection box 14 isadapted to receive and hold a plurality of vials 20 containingbiological materials. Also disposed in or near the vials 20 is aplurality of temperature sensors 25 to be used as inputs to the systemcontroller (not shown). The cryogen intake circuit 12 or conduit isfurther coupled to the gas injection box 14 that is adapted to receivethe cryogen intake flow and evenly distribute the cryogen across theporous metal plate 16. The cold cryogen gas flows in a uniform mannerinto an intake plenum 32 in the gas injection box 14 through the lowerporous metal plate 16 holding the vials 20 into the cooling space 30 andthen to the gas removal box 18 which also includes an upper collectionsurface or area (e.g. shown as the optional porous metal plate 19) andan exhaust manifold 34. From the exhaust manifold 34, the spent nitrogengas exits via the gas exhaust circuit 28 or conduit.

As discussed above, the cooling of the vials 20 is provided by the heattransfer between the vials 20 and the cryogenic cold gas 27 flowingthrough the cooling area 30. The cryogenic cold gas 27 is produced inthe cryogen intake circuit 12 by mixing liquid nitrogen with a warmernitrogen gas or recirculating spent nitrogen gas from the gas exhaustcircuit 28 with appropriate mixing apparatus or valves 36. The vials 20are cooled generally at a slightly slower rate than the cryogenic coldgas. The temperature difference between the vials 20 and the cryogeniccold gas 27 is the thermal driving force to cool down the vials 20.Therefore, it is possible to freeze the vials 20 with any temperatureprofile by precisely controlling the temperature of the cryogenic coldgas 27 at a particular temperature profile.

Preferably, the cryogenic cold gas temperature, and more particularly,the temperature profile is actively controlled in response to theaverage temperatures indicated by the temperature or thermal sensors 25disposed at or near the vials 20. In the present embodiment, the averagetemperatures in a plurality of vials 20 are being used as the inputs forthe active control of the system. Preferably, a cascade based controlmethodology where the system temperatures including vial temperaturesare monitored and controlled by a primary system controller, whichtransfers set point signals and other commands to a slave controllerresponsible for modulating the cryogenic cold gas temperatures in theintake circuit. As discussed in more detail below, the cryogenic coldgas temperature profile is created through the operative control of amixing valve that blends a specified volume of cold liquid nitrogen witha specified volume of warmer nitrogen gas. The blending or mixing ispreferably a continuous operation that changes as a function of time toyield a cryogenic cold gas having a temperature that follows aprescribed temperature profile (i.e. temperature that changes as afunction of time). In short, operative temperature control of theuniform flow cryogenic chiller is achieved by controlling thetemperature profile of the cryogenic cold gas in the intake circuits. Asdiscussed above, it has been found that precise control of the cryogeniccold gas temperatures and temperature gradients has a direct correlationto the observed cooling rates of the given biological material.

In the illustrated embodiment, as the cryogenic cold gas enters thelower gas injection box 14, the cryogenic cold gas 27 is dispersed intoan intake plenum 32 through a series of downward oriented sparger pipesor channels within the gas injection box (not shown). This dispersion inthe intake plenum 32 promotes an even distribution of the cryogenic coldgas 27 across the entire surface of the porous metal plate 16. Thedownward oriented distribution of cryogenic cold gas 27 in the intakeplenum 32 avoids the direct impingement of the cryogenic cold gas 27 onthe porous metal plate 16, resulting in cold spots and non-uniformcooling. The porous metal plate 16 in the gas injection box 14 forcesthe cryogenic cold gas 27 to distribute uniformly across the entirecooling area 30 of the uniform flow cryogenic chiller 10, where thevials or other containers of biological material are held. Alternativemeans or devices for cryogenic cold gas dispersion or distributionacross the entire surface of the porous metal plate are contemplated anduse of the above described downward oriented sparger pipes or channelswithin the gas injection box is an additional example of such gasdispersion or distribution for the present disclosure.

In one illustrated embodiment, the spent nitrogen is collected in anexhaust manifold 34 disposed above the gas collection area or surface(e.g. porous plate 19) in the gas removal box 18. As illustrated, thecryogenic cold gas 27 has only a short path to traverse from the intakeplenum 32 through the porous plate 16 upward into the cooling area 30,through the upper porous plate 19 and into the exhaust manifold 34. Theuniform direction and short distance of the cryogenic cold gas flowresults in a high level of uniformity in vial 20 cooling within thecryogenic chiller 10. Pore sizes for the porous metallic plates 16, 19are preferably on the order of about 2 to 50 microns in diameter, assmall pores enhance the dispersion and resulting uniformity in cooling.By cooling or freezing the biological material at the optimized rate,the survival rate of the cells is enhanced yielding potentially higherdrug potency.

At the freezing point of the solutions, the heat of crystallizationkeeps the solution temperature from dropping, and sometimes thetemperature within the vial can also rise. Using one or more thermal ortemperature sensors 25 embedded in or near selected control vials, thetemperature of cryogenic cold gas can be adjusted to minimizetemperature deviation from the optimized cooling rate, as needed. Inother words, control of the system may be either pre-programmed or maybe a real-time feedback based operation.

Pharmaceutical, biopharmaceutical or biologic solutions contained invials or containers for cryopreservation would benefit from the presentsystem and methods. Such biological or biopharmaceutical materials mayinclude microorganisms, tissues, organs, stem cells, primary cells,established cell lines, small multicellular organisms, complex cellularstructures such as embryos, or a solution or mixture that includes: liveor attenuated viruses; nucleic acids; monoclonal antibodies; polyclonalantibodies; biomolecules; nonpeptide analogues; peptides, proteins,including fusion and modified proteins; RNA, DNA and subclasses thereof,oligonucleotides; viral particles; and similar such materials orcomponents thereof. Also, the containers used for holding the biologicalmaterials may include vials, straws, polymeric bags, or other form ofsuitable container.

FIGS. 3, 4, and 5 depict various embodiments of the present uniform flowcontrolled rate freezer or cryogenic chiller incorporating the uniformflow approach or concept. More specifically, FIG. 3 is a diagram of asingle modular unit 40 of the controlled rate freezer adapted to holdone of the uniform flow cryogenic chillers. The external housing for theunit 40 shown in FIG. 3 is comprised of a solid stainless steel housingwith a gas injection box 44 having an intake conduit 42, a plenum, andporous plate 46 as well as a gas removal box 48 having a porous plate,an exhaust manifold, and an exhaust conduit. The unit shown isdimensioned to hold a single uniform flow cryogenic chiller as describedabove with reference to FIGS. 1 and 2.

FIG. 4 depicts a multi-batch or commercial scale unit 50 that includes acooling chamber 52 that includes a plurality of shelves or rails 54adapted to hold multiple uniform flow cryogenic chiller assemblies. Sucha multi-batch or commercial scale unit 50 is preferably capable ofcryopreserving 50,000 or more vials or other such containers perproduction run. As seen in FIG. 4, the cryogen intake circuit 56 andspent gas exhaust circuit 58 are designed and sized to circulatesufficient cryogen to the multiple individual cryogenic chillers 60.Control system 70 is used to operatively control the temperature profileof the cold cryogen gas provided to each shelf 54, or to each cryogenicchiller assembly 60 depending on the inputs from the thermal sensorsdisposed within the system.

FIG. 5 depicts yet another possible commercial scale embodiment of thecontrolled rate freezer or chiller system 80 that operates in acontinuous or conveyorized manner. Again, the unit 80 and cryogenic coldgas intake circuit 90 and gas exhaust circuit 92 are designed and sizedto circulate sufficient cryogenic cold gas to individualized containersor tray assemblies 88 disposed along a conveyor 86 within thetunnel-type freezer chamber 82 having an entrance and exit means 84. Inthis continuous operation, the cooling profiles of different containers,vials or trays could be either time based, as described above, orspatially based.

The ability to precisely control the cooling rate of biological materialprovides many benefits. For example, biological material frozen in anaqueous solution may experience various stresses during the freezing andsubsequent thawing process that may impair the function or activity ofthe material. Ice formation may physically disrupt the material orcreate severe changes in the interfacial bonding, osmotic forces, soluteconcentrations, etc. experienced by the material. Proper design of thefreezing process can mitigate such stresses and the present system andmethod allows for the precise control of the freezing process to achieveuniformity in the frozen material in all vials in accordance with thedesigned freezing profile.

One exemplary system includes a cryogen source, an intake circuitcoupled to the cryogen source and adapted for providing a uniform flowand temperature of a cryogenic cold gas to a cooling chamber, an exhaustcircuit and a control system. The cooling chamber comprises an intakeplenum, an exhaust manifold, and two or more parallel surfaces thatdefine a cooling area between adjacent parallel surfaces with one of theparallel surfaces (i.e distribution surface) disposed adjacent to theintake plenum and in fluid communication with the intake plenum andanother of the parallel surface (i.e. collection surface) disposedadjacent to the exhaust manifold, the parallel collection surfaces andcooling area adapted to retain, or hold, a plurality of containers ofbiological materials. The exhaust circuit of the freezing or chillingsystem is adapted to remove the cryogen gas from the exhaust manifold ofthe cooling chamber whereas the control system is adapted to adjust theflow rates of the cryogen source in the intake circuit and any cryogengas in the exhaust circuit to adjust the temperature of the cold cryogengas delivered to the cooling chamber in response to a desired coolingrate of the biological materials and measured temperatures within thecooling chamber. In this manner, a uniform, unidirectional flow oftemperature adjusted cryogenic cold gas is delivered to the cooling areabetween the parallel porous surfaces and parallel to each of theplurality of containers to uniformly cool the biological materials.

The operation of the above disclosed exemplary system includes the stepsof: (i) placing a plurality of containers of the biological materials ina cooling area defined as the area between parallel porous surfaceswithin a cooling chamber; (ii) mixing a liquid cryogen with a warmer gasto produce a cold cryogenic gas at a selected temperature profile, thetemperature profile corresponding to a desired cooling rate of thebiological materials within the containers; (iii) delivering aunidirectional flow of the temperature adjusted cryogenic cold gasthrough one of the porous surfaces to the cooling area between theparallel porous surfaces and parallel to each of the plurality ofcontainers to uniformly cool the biological materials; and (iv) promptlyexhausting the gas from cooling chamber via another parallel poroussurface so as to prevent recirculation of the gas within the coolingarea.

Turning now to FIG. 9, a detailed embodiment is presented. Theillustrated cryogenic chiller system 210 includes a cooling chamber 220adapted to receive a cryogenic cold gas 260 from a cryogen cold gascircuit 262, a source of liquid nitrogen 230, a liquid supply circuit232 including a phase separator 234, a supply of gaseous nitrogen 240, agas supply circuit 242, a recirculating cryogenic gas 250 and a gasrecirculation circuit 252. The cryogenic chiller system 210 furtherincludes a programmable logic controller (PLC) based control system 270that operatively controls the fluid circuits in response to measuredtemperatures and pressures as well as certain user defined parametersincluding the desired cooling profiles.

The illustrated cooling chamber 220 has a plurality of cooling shelves222 used to cool a large number of vials containing pharmaceuticalactive ingredients or active biological molecules. A cryogenic cold gas260 is supplied to the cooling chamber 220 from a static in-line mixer263 that mixes liquid nitrogen from the source of liquid nitrogen 230via the liquid supply circuit 232 with a precisely metered gaseousnitrogen gas stream from the gas supply circuit 242 and recirculatingcryogenic gas 250 from the gas recirculation circuit 252.

The temperature of the cryogenic cold gas 260 is preferably measuredwith a temperature sensor 264 disposed downstream of the static in-linemixer 263. By precisely adjusting the flow of nitrogen from the liquidsupply circuit 232 with nitrogen gas from the gas supply circuit 242 andthe gas recirculation circuit 252 it is possible to rapidly shift thetemperature of the cryogenic cold gas 260 which allows cooling of thevials in the cooling chamber 220 with a wide range of cooling profilesto optimize operating conditions and maximize cell viability, biologicalactivity, drug uniformity, as well as drug potency.

Once a cryogenic cold gas 260 is formed by mixing this nitrogen gas withliquid nitrogen, it is split into multiple levels of cooling shelves 222in a single cooling chamber 220. To provide the exact split of thecryogenic cold gas 260 to the multiple cooling shelves 222, a pluralityof critical flow orifices 265 are used to split cryogenic cold gas 260into multiple gas streams. Under critical choke flow conditions, thecryogenic cold gas flow to the cooling shelves 222 is maintainedindependent of the downstream pressure. A large cryogenic cold gasmanifold 266 is used to eliminate or minimize pressure differencesupstream of the critical flow orifices 265 while the downstream gas flowresistance has no impact on the gas flow through the critical floworifices 265. In this manner, the cryogenic cold gas flow to each ofcooling shelves 222 in the cooling chamber 220 is nearly identical.

The cryogenic chiller system 210 is a direct contact cooling system witha cryogenic cold gas 260 flowing in the same direction with respect toeach vial and preferably along the longitudinal axis of the vials, thuscreating a uniform cooling profile for all the vials. A porous metallicmembrane (See FIGS. 1 and 2) provides uniform resistance across all thecooling surfaces, thus allowing the individual vials to receive uniformamounts of refrigeration.

The nitrogen gas supply 240 is preferably received from a bulk storagetank and is directed through a filter 244 to remove particulatematerials. The nitrogen gas supply 240 is then regulated down to thedesired pressure through a discharge pressure regulator 245. Linepressures before and after the pressure regulator 245 are preferablymonitored using one or more pressure indicators 246. A mass flowcontroller 247 including a mass flow sensor 248 with electro-pneumaticcontrol valve 249 is preferably used to control and maintain a preciselymetered nitrogen gas flow rate through the gas supply circuit 242 to thestatic in-line mixer 263. An electrical solenoid valve 243 is alsoincluded in the gas supply circuit 242 to provide positive shut offcapability when the cryogenic chiller system 210 is not operating.Alarms can be set in the control system 270 to deactivate this solenoidvalve 243 if emergency shutdown of the cryogenic chiller system 210 isrequired.

The illustrated system depicts an additional source of gas, namely air,to be used to operate various control valves. The illustrated air supplycircuit 215 includes a filter 216 adapted to remove any particulatesfrom the line, a pressure regulator 218 that is adapted to reduce theair pressure to about 25 psig for safe operation, and one or morepressure indicators 219 used to monitor the pressure in the air supplycircuit 215.

The liquid nitrogen supply circuit 232 includes a source of liquidnitrogen 230, a phase separator 234, one or more temperature andpressure sensors 233, a liquid nitrogen manifold 235, one or moresafety/relief valves 236, a strainer 237, and a primary cryogenic flowcontrol valve 238. All liquid nitrogen piping is preferably insulated soas to minimize any phase change of the liquid nitrogen to nitrogen gasand the resulting two-phase flow in any of the pipes within the liquidnitrogen supply circuit 232.

The liquid nitrogen phase separator 234 is designed to remove anynitrogen gas that forms in the liquid nitrogen supply circuit 232 due toheat leakage or changes in pipeline pressures. The illustrated phaseseparator 234 is a double-walled, vertically mounted, cylindrical tank.The inner liquid vessel has a maximum allowable working pressure (MAWP)rating of 250 psig, with the outer vessel providing a vacuum insulation.The gas phase vent valve 239 operatively controls the filling of thephase separator 234 with liquid nitrogen from the source of liquidnitrogen 230. At a low liquid level, the gas phase vent valve 239 opensto vent 280 vapor pressure from the phase separator 234, allowing liquidnitrogen to transfer from the source of liquid nitrogen 230. As theliquid nitrogen level increases in the phase separator 234, gas phasevent valve 239 begins to close and the fill rate decreases until thevalve 239 is completely closed and filling of the phase separator 234with liquid nitrogen stops.

The strainer 237 is coupled to a blow-down relief valve 236A that isoperated as required to clean the strainer 237 or purge any vaporizednitrogen gases from the liquid nitrogen supply circuit 232. The strainer237 also serves to filter out any particulates in the liquid nitrogen soas to avoid adverse performance or damage to the primary cryogeniccontrol valve or relief valves.

One of the illustrated safety valves is a cryogenic electrical solenoidvalve 236B that provides positive shutoff of the liquid nitrogen supply.Deactivating the electrical solenoid valve 236B shuts off all liquidnitrogen flow through the liquid nitrogen supply circuit and to thestatic in-line mixer 263. This electrical solenoid valve 236B isconfigured such that cutting electrical power immediately stops theliquid nitrogen flow through the liquid nitrogen supply circuit 232circuit and vent 280 any trapped liquid nitrogen from the circuit. Inaddition, other process shutdown and the emergency shutoff procedureswithin the control system 270 generate command signals to the one ormore safety valves 236 as, for example, when the cryogenic chillersystem 210 has stopped operating at the end of the freezing cycle or forother reasons including preset alarm conditions. The control system 270stops the liquid nitrogen flow in the liquid nitrogen supply circuit 232by shutting off one or more of the safety valves 236.

The primary cryogenic flow control valve 238 receive signals from thecontrol system 270 to control the amount of liquid nitrogen supplied tothe cryogenic cold gas circuit 262 in response to measured temperaturesand pressures within the cryogenic chilling system 210 as well ascertain user defined parameters including the desired cooling profiles.

Liquid nitrogen from the liquid nitrogen supply circuit 232 is directedto the static in-line mixer 263. The liquid nitrogen evaporates into acryogenic cold gas 260 by mixing with the nitrogen gas directed from thegas supply circuit 242 and the gas recirculation circuit 252. The staticin-line mixer 263 is used to ensure that no slug of unevaporated liquidnitrogen enters the cooling chamber 220. The temperature in thecryogenic cold gas circuit 262 is monitored with a temperature sensor264 disposed at or near the exit of the static in-line mixer 263. Thecontrol system 270 receives this measured temperature and regulates theliquid nitrogen flow rate and gas flow rates to the static in-line mixer263 in response thereto based on programmed temperature profiles andpreset parameters to adjust the temperature of the cryogenic cold gas.

Downstream of the static in-line mixer 263, the cryogenic cold gas 260is directed to a large cryogenic cold gas manifold 266 and then to themultiple cooling shelves 222 in the cooling chamber 220 via a pluralityof critical flow orifices 265. The large cryogenic cold gas manifold 266is used to ensure that all the gas distribution points realize the sameor similar pressures. The actual cryogenic cold gas flow rate deliveredto each of the cooling shelves 222 of the cooling chamber 220 isdetermined by the size of the critical flow orifice 265 associated witheach cooling shelf 222.

Inside the cooling chamber 220 at each level, there are a series of gasdistribution pipes with downward oriented nozzles. The purpose of theadditional gas distribution pipes inside the cooling chamber is to avoidor minimize velocity generated local pressure gradients that may impactthe cryogenic cold gas distribution across any large porous metallicmembrane. With the critical flow orifices 265 and gas distributionnetworks, a large cooling chamber can be used holding thousands of vialsor packages with very high degree of cooling uniformity.

The cooling surfaces within the multiple levels of the cooling chamber220 are made of porous metallic membranes 227 adapted to generateuniform gas flow across the plurality of vials. Due to the small poresize and high flux in the metallic membranes 227, a generally laminarflow rising from the entire cooling surface is generated. While agenerally laminar flow from the cooling surface is preferred, otherflows, including a turbulent gas flow are tolerable so long as the flowremains generally parallel to the vials and that macro recirculation ofthe gas does not occur inside the cooling chamber 220.

Above the porous metallic membranes at each level in the cooling chamber220 is an exhaust manifold 225 with a perforated plate disposed in aparallel orientation with the porous metallic membranes 227 to maintainthe uniform flow of the cryogenic cold gas 260 during the cooling of thevials. The gas received in the exhaust manifold 225 is immediatelyremoved from the cooling chamber 220 in order to avoid or minimize anyinternally recirculating flow of the spent nitrogen gas. It is importantto avoid the internal recirculation of the nitrogen gas as suchrecirculated gas is generally at a warmer temperature than the cryogeniccold gas 260 supplied to the cooling chamber 220. Such internallyrecirculating flow is the main cause of temperature non-uniformity withedge effects in prior art or conventional cooling devices.

The exhausted gas removed from the cooling chamber 220 is preferablydiverted to a gas recirculation circuit 252. The illustrated gasrecirculation circuit 252 includes a recirculating gas manifold 253disposed between the exhaust manifolds 225 in the cooling chamber 220and a recirculating blower 254 that starts automatically during thelater part of the freezing cycle. The gas recirculation circuit 252 alsoincludes a mass flow meter 255 coupled to the control system 270 thatmeasures the flow through the gas recirculation circuit 252 so as toadjust the make-up gas flow rate from the gas supply circuit 242 tomaintain a desired level of cryogenic cold gas 260 flow in the cryogeniccold gas circuit 262. Back pressure regulator 256 maintains the pressurefrom the recirculating blower 254 while check valve 258 keeps themake-up nitrogen gas from the gas supply circuit 242 from entering thegas recirculation circuit 252 when the recirculation blower 254 is notoperating. Safety relief valve 259 provides over-pressurizationprotection for cooling chamber 220 in case there are blockages in gasrecirculation circuit 252.

The pressure and temperature inside the cooling chamber 220 aremonitored with pressure gauge 228 and temperature sensors 229 orthermocouples disposed within the cooling chamber 220 proximate some ofthe vials. The pressure gauges 228, temperature sensors 229 as well asthe thermocouples are coupled to and provide inputs to the controlsystem 270.

The above disclosed cryogenic chiller system is able to provide uniformtemperatures and flows of cryogenic cold gas to each vial due to thedisclosed unidirectional and uniform flow of cryogenic cold gasgenerally parallel to the longitudinal axis of the vials or containers.In some embodiments, this flow may be approximated as a plug flowthrough cooling chamber 220 delivering a uniform temperature and flowrate of cryogenic cold gas to each of the vials of material.

FIG. 10 presents another embodiment of a cryogenic chiller system 300.In this particular embodiment, a source of liquid cryogen 302 acts asthe supply source for both the liquid cryogen and gas flows in thecryogenic chiller system. The liquid cryogen source may include, but isnot limited to liquid: argon, nitrogen, air, permissible mixturesthereof, and any other appropriate cryogen that is non-reactive with thesystem and material to be cryopreserved. The source of liquid cryogen302 is connected to a safety valve 304. In one embodiment safety valve304 is an electronically controlled solenoid valve. Safety valve 304shuts off the flow of cryogen to the system when the system isde-energized, the pressure in the exhaust system is greater than orequal to the pressure in the cooling chamber, a manual safety switch hasbeen thrown, a preset alarm activates, or other appropriate situations.When safety valve 304 is open liquid cryogen is supplied to phaseseparator 306. Phase separator 306 has two output flows to conduits 308a and 308 b.

The first flow provided from phase separator 306 flows through conduit308 a. This first flow provides liquid cryogen or a mix of liquidcryogen and gas to vaporizer 314. Vaporizer 314 vaporizes any liquidcryogen present in the flow to create a flow of gas. The vaporizer isconnected to a pressure relief valve 316 and heater 318. In a preferredembodiment, pressure relief valve 316 is set to 100 psi (gauge). Heater318 heats the gas flow to adjust the temperature to that indicated bythe control systems. Temperature sensor 324 monitors the temperature ofthe gas flow exiting heater 318 and outputs a signal to temperaturecontroller 326. Temperature controller 326 actuates a relay 320 tocontrol heater 318. In a preferred embodiment relay 320 is a solid staterelay. However, relay 320 may be replaced with a circuit or othersuitable component capable of receiving instructions from temperaturecontroller 326 and controlling heater 318. The flow of temperatureadjusted gas is provided from heater 318 through safety valve 322 toflow control valve 332. A flow sensor 330 monitors the flow oftemperature adjusted gas and outputs a signal to flow controller 328.Flow controller 328 controls flow control valve 332 which in turncontrols the flow rate of temperature adjusted gas provided to mixer334.

The second flow provided from phase separator 306 is liquid cryogenflowing through conduit 308 b. The flow of liquid cryogen is directedthrough flow control valve 310. Flow control valve 310 controls the flowrate of liquid cryogen. The flow of liquid cryogen is combined with theflow of temperature adjusted gas provided to mixer 334. All conduits,valves, and control systems associated with transporting the liquidcryogen are preferably insulated to minimize any unwanted phase changeof the liquid cryogen to a gas.

The separate flows of temperature adjusted gas from flow control valve332 and liquid cryogen from flow control valve 310 are mixed together inmixer 334 to provide cryogenic cold gas. In some embodiments, mixer 334may be a static mixer, an arrangement of valves, an impeller, or anyother appropriate structure adapted for mixing the flows. Thetemperature of the cryogenic cold gas is monitored by temperature sensor336 after exiting mixer 334. Temperature sensor 336 provides a signal totemperature controller 312. Temperature controller 312 adjusts the flowof cryogen through flow control valve 310 so as to adjust thetemperature of the cryogenic cold gas exiting mixer 334.

The flow of cryogenic cold gas is provided to cooling chamber 338holding a plurality of vials or containers. The cryogenic cold gas flowsthrough the chamber so as to transfer heat with the vials or containers.To provide uniform cooling of each vial or container, the cryogenic coldgas is uniformly distributed throughout cooling chamber 338 by at leastone gas injection box 340. The cryogenic cold gas is immediatelyexhausted from the cooling chamber by at least one gas exhaust box 342preferably arranged above the at least one gas injection box 340. Thecryogenic cold gas is immediately exhausted to avoid any recirculationof the cryogenic cold gas since this will lead to non-uniformities inthe cooling chamber's 338 temperature. Cooling chamber 338 is furtherprovided with pressure relief valve 346 preferably set at 50 psig.Pressure sensor 348 monitors the pressure in cooling chamber 338. Asignal is output from pressure sensor 348 to liquid nitrogen controller312, gas flow controller 328, and pressure controller 350. Pressurecontroller 350 controls flow control valves 354 and 358. In someembodiments, the system may reduce or shut down the liquid and gas flowsduring pressurization and depressurization steps in response to a signalfrom pressure sensor 348.

In a preferred embodiment, porous membranes 344 suitable for cryogenicuse are attached to the gas injection box 340 and gas exhaust box 342.The porous membranes 344 are adapted to generate a uniform flow of gasacross the plurality of vials. Due to the small pore size and high fluxacross the porous membranes 344, a uniform flow rising from the entirecooling surface is generated. In a preferred embodiment, porousmembranes 344 are porous metallic plates. While a laminar flow from thecooling surface is preferred, a turbulent gas flow or other gas flow istolerable so long as the flow remains parallel to the vials and thatmacro recirculation of the gas does not occur inside the coolingchamber. While the flow is disclosed as flowing upwards it should beunderstood that the flow of gas may be oriented in any direction so longas each of the vials receives a uniform flow and temperature ofcryogenic cold gas.

The cryogenic cold gas exhausted through the at least one exhaust hood342 flows through conduit 352 to adjustable pressure control valve 354prior to being exhausted to system exhaust 362. Adjustable pressurecontrol valve 354 enables pressurization of cooling chamber 338. Thepressure of the exhausted cryogenic cold gas is monitored by pressureswitch 360. Pressure switch 360 actuates safety valves 304 and 322 whenthe pressure of the exhausted cryogenic cold gas is lower than thepressure in cooling chamber 338. Closing safety valves 304 and 322 stopsthe flows of liquid and gas into the system thus preventing nitrogenleakage due to abnormal system operation.

Cooling chamber 338 further includes an adjustable flow control valve356 connected to flow control valve 358. Adjustable flow control valve356 may be adapted for either manual or electronic adjustment. In onepossible embodiment, flow control valve 358 is an on/off control valveactuated by a pneumatic solenoid. In other alternative embodiments, flowcontrol valve 358 may be actuated by an electrical solenoid, anelectrical switch, a pneumatic control system, a hydraulic controlsystem, or any other appropriate mechanism. When open, flow controlvalve 358 enables the depressurization of cooling chamber 338 to systemexhaust 362. The rate of depressurization is controlled by the settingof adjustable flow control valve 356.

In a preferred embodiment, at least a portion of the cryogenic cold gasexhausted through system exhaust 362 is recycled into the system. Therecycled gas is preferably input into the gas flow between vaporizer 314and heater 318 depicted as recycled exhaust gas input 364. However, itis possible to place the recycled exhaust gas input 364 in otheralternative positions within the system.

It should be understood that the separate valves and systems depictedherein may be powered and controlled in a variety of ways. Possiblemethods of providing power and control include, but are not limited to,manual, electrical, pneumatic, and hydraulic control. In addition,cryogenic chiller system 300 may implement any combination of the abovemethods to provide power and control to the separate components. Theselection and application of these different control and power methodsmerely represents a design choice and can be easily implemented by oneof skill in the art.

While the above described methods and systems have been shown withregards to providing uniform temperature profiles during a freezingprocess, the same methods and systems may be applied to provide auniform temperature profile for a plurality of vials or containers foruniformly thawing the material in each of the plurality of vials orcontainers. In both a uniform freezing and thawing process, each vial orcontainer sees substantially the same temperature profile regardless ofits location within the cooling chamber. Similar to providing a uniformtemperature profile during freezing, uniform thawing of a plurality ofvials or containers will result in more repeatable uniform properties.Such properties include, but are not limited to, cell viability,functionality, and/or biological activity. Depending upon the type ofbiological material present in each container or vial, the biologicalmaterial is revived during the thawing process. Reviving the biologicalmaterial entails returning the biological material to its original stateprior to the freezing process. Furthermore, while such systems aretypically used for one type of material at a time it is possible tofreeze multiple biological materials at once inside the system.Therefore, it is possible that at least two of the containers inside ofthe system would contain different biological materials during afreezing process. Such a use is considered within the scope of thisdisclosure.

The above disclosed systems and methods are particularly well-suited forcommercial type or large scale biological production operations sincethe process is conducted using the same equipment and process parametersthat are easily scaled or adapted to manufacture a wide range ofbiological products. The presently disclosed process and system providesfor the controlled rate freezing of biological materials using a processthat achieves a high degree of uniformity in cooling or freezing of thebiological material from sample to sample, vial to vial, container tocontainer, and batch to batch.

Nucleation Control

In addition to the temperature profile seen by each vial or container,the final uniformity of properties and structure from sample to sample,vial to vial, container to container, and batch to batch may also dependon the nucleation temperature. As stated above, this variability in thenucleation temperature can impact properties such as cell activity andviability as well as the crystal structure of the frozen material andthe time needed to complete a freeze drying process. Advantageously, thecurrently disclosed systems and processes may be applied to providecontrol over the nucleation temperature of the material in the pluralityof vials or containers using two possible methods. The methods include,but are not limited to, pressure control induced nucleation andtemperature quench induced nucleation, although nucleation control neednot be used in all embodiments with respect to improving or otherwiseenhancing cell viability and/or other biological activity. For example,substantially uniform cooling features of aspects of the invention maybe used to freeze materials in a plurality of vessels without the use ofnucleation control, yet still provide uniform and enhanced viabilityand/or biological activity features for the materials. Also, it shouldbe understood that enhanced viability and/or biologic activity providedby aspects of the invention may vary according to the materials beingfrozen or otherwise processed, and may be measured in relation to priortechniques.

For example, when working with a first set of materials, priortechniques may be capable of achieving cell viability and/or biologicalactivity in an 80% range, whereas viability and/or biological activityof only 50% may be achievable with a second set of materials. Thus,aspects of the invention may provide enhancements to viability and/orbiological activity of 85% or more for the first set of materials, whichis an improvement over prior techniques, and enhancements to viabilityand/or biological activity of 55% or more for the second set ofmaterials, which likewise provides an improvement over prior techniqueseven though a 55% viability and/or biological activity for the secondset of materials is less than the 80% viability and/or biologicalactivity provided by prior techniques for the first set of materials.The above disclosed processes and systems may also be modified to applyany other appropriate nucleation method including, use of additives, icefog, vial pretreatment, vibration, and vacuum freezing.

Nucleation is the onset of a phase transition in a small region of amaterial. For example, the phase transition can be the formation of acrystal from a liquid. The crystallization process (i.e., formation ofsolid crystals from a solution) often associated with freezing of asolution starts with a nucleation event followed by crystal growth.

In the crystallization process, nucleation is the step where selectedatoms and/or molecules dispersed in the solution or other material startto gather to create clusters in the nanometer scale so as to becomestable under the current operating conditions. These stable clustersconstitute the nuclei. The clusters need to reach a critical size inorder to become stable nuclei. Such critical size is usually dictated bythe operating conditions such as temperature, contaminants, degree ofsupersaturation, etc. and can vary from one sample of the solution toanother. It is during the nucleation event that the atoms in thesolution arrange in a defined and periodic manner that defines thecrystal structure.

Crystal growth is the subsequent growth of the nuclei that succeed inachieving the critical cluster size. Depending upon the conditionseither nucleation or crystal growth may predominate over the other, andas a result, crystals with different sizes and shapes are obtained.Control of crystal size and shape constitutes one of the main challengesin industrial manufacturing, such as for pharmaceuticals.

In addition to temperature control, the present methods and systems maybe used for controlling the time and/or temperature at which a nucleatedphase transition occurs in a material. In freezing applications, theprobability that a material will spontaneously nucleate and beginchanging phase is related to the degree of sub-cooling of the materialand the absence or presence of contaminants, additives, structures, ordisturbances that provide a site or surface for nucleation.

The freezing or solidification step is particularly important incryopreservation and freeze-drying processes where existing techniquesresult in nucleation temperature differences across a multitude of vialsor containers. The nucleation temperature differences tend to produce anon-uniform product and an excessively long drying time forfreeze-drying processes. The present methods, on the other hand, providea higher degree of process control in batch solidification processes(e.g., freeze-drying) and produce a product with more uniform structureand properties Unlike some of the prior art techniques to inducenucleation, the present methods require minimal equipment andoperational changes for implementation.

In principle, the present methods can be applied to any materialprocessing step that involves a nucleated phase transition. Examples ofsuch processes include the freezing of a liquid, crystallization of icefrom an aqueous solution, crystallization of polymers and metals frommelts, crystallization of inorganic materials from supersaturatedsolutions, crystallization of proteins, artificial snow production,deposition of ice from vapor, food freezing, freeze concentration,fractional crystallization, cryopreservation, or condensation of vaporsto liquids. From a conceptual standpoint, the present methods may alsobe applied to phase transitions such as melting and boiling.

The presently disclosed methods represent an improvement to currentpharmaceutical cryopreservation and lyophilization processes. Forexample, within a large industrial system there can be over 100,000vials containing a pharmaceutical product that needs to be frozen and/ordried. Current practice in the industry is to cool the solution to avery high degree so that the solution in all vials or containers in thefreeze-dryer are guaranteed to freeze. However, as discussed above, thenon-uniform cooling and lack of a uniform and consistent nucleationcontrol method, the contents of each vial or container freezes randomlyover a range of temperatures below the freezing point.

Turning now to a closer examination of FIGS. 6-8, the plottedtemperature profiles illustrate that the presently disclosed freezing orchilling process and system can be used to initiate and control thenucleation of freezing in materials using a temperature quench. Asillustrated in FIGS. 6-8, the nucleation of freezing of the materials inall vials monitored occurred at roughly the same time and sametemperature. Nucleation of freezing is exhibited by the concurrent shortspike in sample temperature (see 100, 110, 120) as a result of theexothermic process occurring during the phase change occurring in thesamples. Thus, nucleation control is possible by precisely controllingthe timing and magnitude of a sharp or rapid temperature quench usingthe above described controlled freezing systems and methods.Alternatively, a temperature quench may be referred to as a temperaturespike or cold spike in reference to the same physical process describedabove. In certain embodiments the change in temperature during thetemperature quench may be a step-wise change in temperature or it maychange at a predetermined rate.

In a broad sense, the presently disclosed methods for inducingnucleation of a phase transition within a material via temperaturequench nucleation control comprise the steps of: (i) uniformly coolingthe material to a temperature near or below a phase transitiontemperature of the material; and (ii) uniformly and rapidly decreasingthe temperature of the cryogenic cold gas to induce nucleation of thematerial. Each of these important steps will be discussed in more detailbelow.

FIG. 11 presents a more detailed exemplary temperature profile of thecryogenic gas during a freezing process using a temperature quench tocontrol nucleation. The temperature profile versus time comprises anequilibrium step 402, a cooling step 404, a pre-nucleation temperaturestep 406, a temperature quench step 408, a temperature quench hold step410, a post-nucleation temperature hold step 412, and a final coolingstep 414.

During equilibrium step 402 each of the vials or containers in thesystem are brought to a uniform equilibrium temperature near or belowthe freezing point (i.e. the phase transition temperature) of thematerial prior to beginning the remaining steps in the freezing process.After each vial or container reaches a uniform temperature, the vials orcontainers are further cooled by decreasing the cryogenic cold gas to apre-nucleation temperature during steps 404 and 406.

The change in temperature of the cryogenic cold gas may decreaselinearly as indicated in step 404 and then hold steady as shown in step406, or alternatively the temperature of the cryogenic cold gas maystepwise change to the pre-nucleation temperature indicated in step 406without the need for the linear, or optionally non-linear, temperaturechange shown in step 404. Furthermore, the cryogenic cold gas may beheld at the pre-nucleation temperature for a predetermined time asindicated in step 406 to ensure all vials or containers reach a uniformtemperature prior to nucleating the phase change. Optionally, steps 404and 406 may be performed dynamically without the hold time shown in step406.

The material is nucleated in temperature quench step 408. During step408, the temperature of the cryogenic cold gas is adjusted to atemperature sufficiently low to ensure nucleation of the material ineach vial or container. In a preferred embodiment, the temperatureadjustment of the cryogenic cold gas is sufficiently fast such that thetemperature of the material in each of the vials or containers does notsubstantially change during the temperature adjustment. After nucleatingthe material in each vial or container an optional temperature quenchhold may be applied at step 410.

After performing temperature quench hold step 410 the temperature of thecryogenic cold gas is subsequently raised to a temperature below thefreezing point of the material and held for a set amount of time duringthe post-nucleation temperature hold step 412 to ensure uniformtemperature. After step 412, the cryogenic cold gas is cooled at apredetermined rate to a final desired temperature during final coolingstep 414.

FIG. 12 depicts the temperature profiles 416 of six separate vials andthe cryogenic cold gas temperature profile 418 during a freezing processimplementing temperature quench induced nucleation control. Thecryogenic cold gas profile 418 included a pre-nucleation temperature of−5° C., a quench temperature of −80° C., a post nucleation temperatureof −35° C., a post-nucleation hold time of 10 minutes, and apost-nucleation cooling rate of 2.5° C./min. In contrast FIG. 13 depictsthe temperature profiles 420 of six separate vials and the cryogeniccold gas temperature profile 422 during a freezing process withoutnucleation control. The cryogenic cold gas profile 422 had a coolingrate of 5° C./min. As indicated by the sharp increase in temperaturealong each temperature profile, the samples that underwent the freezingprocess with nucleation control exhibit nucleation temperatures andtimes in a narrower range than those in the freezing process withoutnucleation control. Consequently, the freezing process with nucleationcontrol enables more uniform and repeatable sample temperature profiles.

Further examples of specific temperatures and cooling rates arediscussed in detail below with regards to cell viability testing ofnormal dermal human fibroblast cells during cryogenic preservation.

When compared to the wide spectrum of times and temperatures in thenucleation of freezing that results from use of conventional controlledrate freezers, the present system and method applying nucleation controlvia a temperature quench provides a greater degree of control whichlikely impacts other performance aspects and characteristics of thepreserved biological material. Also, as the contemplated nucleationinitiation and control is temperature driven, it works equally well inopen or closed containers or vials.

As shown above in FIG. 10, pressure control systems may be included topermit pressurization and depressurization control of the system andfreezing process in addition to uniform temperature control. Therefore,in addition to controlling the nucleation temperature via a temperaturequench method, the nucleation temperature may be controlled usingpressure induced nucleation. FIG. 14 depicts a temperature versus timeplot of six vials of an aqueous solution undergoing a conventionalstochastic nucleation process while in a conventional freeze dryer usinga cold shelf arrangement. The plot shows the typical range of nucleationtemperatures of the solution within the vials (511,512,513,514,515, and516). As seen therein, the vial contents have a thermodynamic freezingtemperature of about 0° C., yet the solution within each vial naturallynucleates over the broad temperature range of about −7° C. to −20° C. ormore, as highlighted by area 518. Plot 519 represents the shelftemperature inside the freeze-drying chamber.

Conversely, FIG. 15 and FIG. 16 depict temperature versus time plots ofa solution undergoing a freezing process with depressurized nucleationin accordance with the present methods. In particular, FIG. 15 shows thetemperature versus time plot of six vials of an aqueous solutionundergoing an equilibrated cooling process (See Example 2) withnucleation induced via depressurization of the chamber(521,522,523,524,525, and 526). The vial contents have a thermodynamicfreezing temperature of about 0° C. yet the solution within each vialnucleates at the same time upon depressurization and within a verynarrow temperature range (i.e., −4° C. to −5° C.) as seen in area 528.Plot 529 represents the shelf temperature inside the freeze-dryingchamber and depicts an equilibrated freezing process, one where thetemperature of the shelves is held more or less steady prior todepressurization.

Similarly, FIG. 16 shows the temperature versus time plot of three vialsof an aqueous solution undergoing a dynamic cooling process (See Example7) with nucleation induced via depressurization of the chamber (531,532,and 533). Again, the vial contents have a thermodynamic freezingtemperature of about 0° C. yet the solution within each vial nucleatesat the same time upon depressurization at a temperature range of about−7° C. to −10° C., as seen in area 538. Plot 539 represents the shelftemperature inside the freeze-drying chamber and generally depicts adynamic cooling process, one where the temperature of the shelves isactively lowered during or prior to depressurization.

As illustrated in FIGS. 14-16, the present pressure induced nucleationmethod provides improved control of the nucleation process by enablingthe freezing of solutions to occur within a more narrow temperaturerange (e.g., about 0° C. to −10° C.) and/or concurrently, therebyyielding a product with greater uniformity from vial-to-vial. While notdemonstrated, it is foreseeable that the induced nucleation temperaturerange may even extend slightly above the phase transition temperatureand may also extend to about 40° C. of sub-cooling.

In a broad sense, the presently disclosed methods for inducingnucleation of a phase transition within a material via pressure controlcomprise the steps of: (i) uniformly cooling the material to atemperature near or below a phase transition temperature of thematerial; and (ii) rapidly decreasing the pressure to induce nucleationof the material. Each of these important steps will be discussed in moredetail below.

Step 1—Cooling the Material

Illustrative materials useful in the present method include puresubstances, gases, suspensions, gels, liquids, solutions, mixtures, orcomponents within a solution or mixture. Suitable materials for use inthe present method may include, for example, pharmaceutical materials,biopharmaceutical materials, foodstuffs, chemical materials, and mayinclude products such as wound-care products, cosmetics, veterinaryproducts and in vivo/in vitro diagnostics related products and the like.When the material is a liquid, it may be desirable to dissolve gasesinto the liquid. Liquids in a controlled gas environment will generallyhave gases dissolved in them.

Other illustrative materials useful in the present method includebiological or biopharmaceutical material such as tissues, organs andmulti-cellular structures. For certain biological and pharmaceuticalapplications, the material may be a solution or mixture that includes: alive or attenuated viruses; nucleic acids; monoclonal antibodies;polyclonal antibodies; biomolecules; nonpeptide analogues; peptides,including polypeptides, peptide mimetics and modified peptides;proteins, including fusion and modified proteins; RNA, DNA andsubclasses thereof oligonucleotides; viral particles; and similar suchmaterials or components thereof.

Pharmaceutical or biopharmaceutical solutions contained in vials orcontainers for freeze-drying would be a good example of a material thatwould benefit from the present method. The solutions are mostly waterand are substantially incompressible. Such pharmaceutical orbiopharmaceutical solutions are also highly pure and generally free ofparticulates that may form sites for nucleation. Uniform nucleationtemperature is important to creating a consistent and uniform icecrystal structure from vial to vial or container to container. The icecrystal structure developed also greatly affects the time required fordrying during a freeze drying process.

As applied to a freeze-drying process, the material is preferably placedin a chamber, such as a freeze-drying chamber. Preferably, the chamberis configured so as to allow control of the temperature, pressure, andgas atmosphere within the chamber. The gas atmosphere may include, butis not limited to: argon, nitrogen, helium, air, water vapor, oxygen,carbon dioxide, carbon monoxide, nitrous oxide, nitric oxide, neon,xenon, krypton, methane, hydrogen, propane, butane, and the like,including permissible mixtures thereof. The preferred gas atmospherecomprises an inert gas, such as argon, at a pressure between about 7 toabout 50 psig or more. Temperatures within the freeze-dryer chamber areoften dictated by the freeze-drying process and are easily controlledvia the use of a heat transfer fluid that cools or warms the shelveswithin the chamber to drive the temperature of the vials or containersand the material within each vial or container.

In accordance with the present methods, the material is cooled to atemperature near or below its phase transition temperature. In the caseof an aqueous based solution undergoing a freeze-drying process, thephase transition temperature is the thermodynamic freezing point of thesolution. Where the solution reaches temperatures below thethermodynamic freezing point of the solution, it is said to besub-cooled. When applied to a freezing process of an aqueous-basedsolution, the present method is effective when the degree of sub-coolingranges from near or below the phase transition temperature up to about40° C. of sub-cooling, and more preferably between about 3° C. ofsub-cooling and 10° C. of sub-cooling. In some of the examples describedbelow, the present method of inducing nucleation works desirably evenwhere the solution has only about 1° C. of sub-cooling below itsthermodynamic freezing point.

Where the material is at a temperature below its phase transitiontemperature, it is often referred to as being in a metastable state. Ametastable state is an unstable and transient, but relativelylong-lived, state of a chemical or biological system. A metastablematerial temporarily exists in a phase or state that is not itsequilibrium phase or state. In the absence of any changes in thematerial or its environment, a metastable material will eventuallytransition from its non-equilibrium state to its equilibrium state.Illustrative metastable materials include super-saturated solutions andsub-cooled liquids.

A typical example of a metastable material would be liquid water atatmospheric pressure and a temperature of −10° C. With a normal freezingpoint of 0° C., liquid water should not thermodynamically exist at thistemperature and pressure, but it can exist in the absence of anucleating event or structure to begin the ice crystallization process.Extremely pure water can be cooled to very low temperatures (−30° C. to−40° C.) at atmospheric pressure and still remain in the liquid state.Such sub-cooled water is in a non-equilibrated thermodynamicallymetastable state. It only lacks a nucleation event to cause it to beginthe phase transition whereby it will return to equilibrium.

As discussed above, the present methods of inducing nucleation of aphase transition within a material or freezing a material can beutilized with various cooling profiles, including, for example, anequilibrated or a dynamic cooling environment (See FIGS. 15 and 16).

Step 2—Rapidly Decreasing the Pressure

When the material has reached the desired temperature near or below thephase transition temperature, the chamber is quickly or rapidlydepressurized. This depressurization triggers the nucleation and phasetransition of the solution within the vials or containers. In thepreferred embodiment, chamber depressurization is accomplished byopening or partially opening a large control valve that separates thehigh pressure chamber from either the ambient environment or a lowerpressure chamber or environment. The elevated pressure is quicklylowered by mass flow of gas atmosphere out of the chamber. Thedepressurization needs to be fairly fast to induce the nucleation. Thedepressurization should be finished in several seconds or less,preferably 40 seconds or less, more preferably 20 seconds or less, andmost preferably 10 seconds or less.

In typical freeze-drying applications, the pressure difference betweenthe initial chamber pressure and the final chamber pressure, afterdepressurization, should be greater than about 7 psi, although smallerpressure drops may induce nucleation in some situations. Most commercialfreeze-dryers can readily accommodate the range of pressure drops neededto control nucleation. Many freeze-dryers are designed with pressureratings in excess of 25 psig to withstand conventional sterilizationprocedures employing saturated steam at 121° C. Such equipment ratingsprovide an ample window to induce nucleation following protocols thatdepressurize from starting pressures above ambient pressure or thepressure in the surrounding environment. The elevated pressure andsubsequent depressurization can be achieved through any known means(e.g., pneumatic, hydraulic, or mechanical). In the preferredembodiments, operating pressures for the present methods should remainbelow the supercritical pressure of any applied gas, and subjecting thematerial to extreme low pressures (i.e., about 10 mTorr or less) shouldbe avoided during nucleation of the material.

While not wishing to be bound to any particular mechanism, one possiblemechanism to explain the controlled nucleation observed in the practiceof the presently disclosed depressurized nucleation method is that gasesin solution in the material come out of solution upon depressurizationand form bubbles that nucleate the material. An initial elevatedpressure increases the concentration of dissolved gas in the solution.The rapid decrease in pressure after cooling reduces the gas solubility,and the subsequent release of gas from the sub-cooled solution triggersnucleation of the phase transition.

Another possible mechanism is that the temperature decrease of the gasproximate the material during depressurization causes a cold spot on thesurface of the material that initiates nucleation. Another possiblemechanism is that the depressurization causes evaporation of some liquidin the material and the resultant cooling from the endothermicevaporation process may initiate the nucleation. Another possiblemechanism is that the depressurized cold gas proximate the materialfreezes some vapor either in equilibrium with the material prior todepressurization or liberated from the material by evaporation duringdepressurization; the resultant solid particles re-enter the materialand act as seeds or surfaces to initiate nucleation. One or more ofthese mechanisms may contribute to initiation of nucleation of freezingor solidification to differing extents depending on the nature of thematerial, its environment and the phase transition being nucleated.

The process may be carried out entirely at a pressure greater thanambient pressure or over a range of pressures spanning ambient pressure.For example, initial chamber pressure can be above ambient pressure andthe final chamber pressure, after depressurization, can be above ambientpressure but less than the initial chamber pressure; the initial chamberpressure can be above ambient pressure and the final chamber pressure,after depressurization, can be about ambient pressure or slightly belowambient pressure. In addition, the chamber pressure may be increased,prior to depressurization, while uniformly cooling the samples or thestep of increasing the pressure may be carried out in a separatedistinct step.

The rate and magnitude of the pressure drop are also believed to be animportant aspect of the present methods. Experiments have shown thatnucleation will be induced where the pressure drop (ΔP) is greater thanabout 7 psi. Alternatively, the magnitude of the pressure drop may beexpressed as an absolute pressure ratio, R═P_(i)/P_(f), where P_(i) isinitial absolute pressure and P_(f) is final absolute pressure. It isbelieved that nucleation may be induced upon depressurization where theabsolute pressure ratio, R, is greater than about 1.2 in many practicalapplications of the present methods. The rate of pressure drop alsoplays an important role in the present methods. One method ofcharacterizing the rate of pressure drop is through use of a parameter,A, where A=ΔP/Δt. Again, it is surmised that nucleation will be inducedfor values of A greater than a prescribed value, such as about 0.2psi/sec. Empirical data through experimentation should aid one toascertain the preferred pressure drop and rate of pressure drop.

The above disclosed method of inducing nucleation via pressure controlmay be implemented in a conventional freeze-dryer or the currentlydisclosed uniform flow cryogenic chiller. Turning now to FIG. 17, afreeze-dryer is illustrated as freeze-dryer unit (600) which has variousmain components plus additional auxiliary systems to carry out alyophilization cycle. In particular, the freeze-dryer unit (600)includes a lyophilization chamber 602 that contains the shelves 604adapted to hold vials or containers of the solution to be lyophilized(not shown). The solution to be lyophilized is specially formulated andtypically contains the active ingredient, a solvent system and severalstabilization agents or other pharmaceutically acceptable carriers oradditives. Lyophilization of this formulation takes place fromspecialized containers located on hollow shelves. These containers mayinclude vials with stoppers, ampoules, syringes, or, in the case of bulklyophilization, pans.

The illustrated freeze-dryer unit 600 also includes a condenser 606 thatis adapted to remove the sublimated and desorbed solvent from the vaporphase by condensing or freezing it out as ice to maintain adequatevacuum inside the freeze-dryer. The condenser 606 can be internallylocated in the lyophilization chamber 602 or as a separate external unitin communication with the lyophilization chamber 602 through a so-calledisolation valve. The freeze-dryer unit 600 also preferably includes avacuum pump 608 operatively coupled to the condenser 606 and adapted topull a vacuum on lyophilization chamber 602 and condenser 606.

The cryogenic refrigeration system 610 provides the temperature controlmeans for the freeze-dryer unit 600 by cooling a prescribed heattransfer fluid which is circulated to the shelves 604 within thelyophilization chamber 602 and the condenser 606. As illustrated, thecryogenic refrigeration system 610 comprises a source of cryogen 618,such as liquid nitrogen, a cryogenic heat exchanger 620, and a heattransfer fluid circuit 622, a vent 624, a heater 626 and pumps 627,628.

The cryogenic heat exchanger 620 is preferably an NCOOL™ Non-FreezingCryogenic Heat Exchange System available from Praxair, Inc. An importantaspect of the cryogenic heat exchanger 620 is the vaporization of theliquid nitrogen within or internal to the heat exchanger yet in a mannerthat avoids direct contact of the liquid nitrogen on cooling surfacesexposed to the heat transfer fluid. Details of the structure andoperation of such a heat exchanger can be found in U.S. Pat. No.5,937,656; the disclosure of which is incorporated by reference herein.

The prescribed heat transfer fluid circuit 622 is adapted to circulate aheat transfer fluid and is operatively coupled to both thelyophilization chamber 602 as well as the condenser 606. Morespecifically, the heat transfer fluid circulates inside the hollowshelves 604 within the lyophilization chamber 602 to preciselycommunicate the cooling or heating through the shelves 604 to thesolution as needed. In addition the prescribed heat transfer fluid alsoflows through the condenser 606 to provide the cooling means necessaryto sublimate the ice and further desorb the solvent.

Pump 627 and heater 626 are disposed along the heat transfer fluidcircuit 622 upstream of the lyophilization chamber 602 and downstream ofthe cryogenic heat exchanger 620. The pump 627 is sized to move the heattransfer fluid through the heat transfer circuit 622 at the requiredflow rates. The heater 626 is preferably an electric heater adapted toprovide supplemental heat to the heat transfer fluid and thelyophilization chamber 602 as may be required during the dryingprocesses.

As seen in the embodiment of FIG. 17, the condenser 606 is also cooledby a recirculation low temperature heat transfer fluid. Refrigeration ofthe heat transfer fluid flowing through the condenser 606 is alsoprovided by a cryogenic heat exchanger 620. The cryogenic heat exchanger620 is capable of cooling heat transfer fluid continuously withoutfreezing. During the drying phases, the cryogenic heat exchanger 620 isset or adapted to achieve the lowest temperature required for thecondenser 606. As described above, the cryogenic heat exchanger 620pre-evaporates liquid nitrogen into a cryogenic cold gas for heattransfer to the heat transfer fluid. Through pre-evaporation of theliquid nitrogen, this assures the liquid nitrogen avoids boiling offdirectly over a heat exchange surface where the heat transfer fluid isdisposed on the other side. Such an arrangement avoids freezing of thecryogenic heat exchanger 620 since liquid nitrogen boils at about −195degrees Centigrade at atmospheric pressure.

The illustrated embodiment of FIG. 17 also includes a means forcontrolling the gas atmosphere of the lyophilization chamber 650, and inparticular the gas composition and pressure within the chamber 602.Controlling the pressure of the chamber 602 allows for thepressurization and rapid depressurization of the chamber to inducenucleation of the solution. The disclosed embodiment preferably uses oneor more flow control valves 652 controllably adapted to facilitate theintroduction of a pressurized gas atmosphere to the chamber 602 from asource of gas (not shown) and to depressurize the chamber by venting thepressurized gas atmosphere away from the chamber 602 in a controlled andpreferably rapid manner thereby inducing the nucleation of the solutionin the various containers or vials.

Although not shown, the freeze-dryer unit 600 also includes variouscontrol hardware and software systems adapted to command and coordinatethe various parts of the freeze-drying equipment, and carry out thepre-programmed lyophilization cycle. The various control hardware andsoftware systems may also provide documentation, data logging, alarms,and system security capabilities as well. In addition, auxiliary systemsto the freeze-dryer unit 600 may include various subsystems to clean andsterilize the lyophilization chamber 602, auto-load and unload theproduct in the lyophilization chamber 602; and associated cryogenicsystem accessories such as refrigeration skids, liquid nitrogen tanks,piping, valves, sensors, etc. Furthermore, the freeze-dryer unit 600 maybe used optionally with or include the currently disclosed uniform flowcontrolled rate freezer system to cool and freeze the vials orcontainers in the system prior to initiating the freeze-drying process.

FIG. 18 a presents a plot of an illustrative temperature and pressureprofile versus time of the cryogenic cold gas during a depressurizednucleation freezing process as might be applied in the above disclosedcryogenic chiller, freeze-dryer unit, or other appropriate system.During equilibrium step 702 all vials in the chamber are brought to thesame temperature prior to further cooling. During equilibrium step 702the cooling chamber is preferably not actively pressurized. However theinvention is not limited in this regards, in certain embodiments thecooling chamber may be pressurized above or below atmospheric pressure.After equilibrating all of the vials or containers to the sametemperature the cryogenic cold gas is cooled at a predetermined rate tothe pre-nucleation temperature (PNT) and held for a selected amount oftime during steps 704 and 706. The vials cool in response to thedecreased temperature of the cryogenic cold gas and approach the PNT.While the cryogenic cold gas is held at the PNT a pressurization anddepressurization step 708 is applied. During pressurization anddepressurization step 708, the chamber pressure is increased and heldfor a predetermined amount of time prior to depressurizing the system.Nucleation is induced by the depressurization. Alternatively, the priorsteps could be conducted at a first pressure and nucleation can beinduced by depressurizing the system to a lower second pressure withoutthe need to pressurize the system. After nucleation is induced, thecryogenic cold gas is further cooled during step 710 to a desired finaltemperature (i.e. −80° C.) at various rates. Without wishing to be boundby theory it is believed that the nucleation control is relativelyinsensitive to the rate of pressurization. However, it is necessary toensure that a sufficient pressure drop occurs over a short enough timeperiod to induce nucleation. Furthermore, it will be necessary todetermine optimal conditions for each new material to be frozen.Examples of testing regarding the above discussed nucleation process isprovided below.

FIG. 18 b presents temperature and pressure profiles for samplesundergoing the process described above in connection with FIG. 18 a.Four separate 2 ml vials were filled with 1 ml of phosphate bufferedsaline and 10 (v/v) % Dimethyl sulfoxide (DMSO) solution. The four vialswere then placed in randomly selected spaced apart locations on a tray.The tray was then placed into a uniform flow cryogenic chiller withpressure control. The temperatures of the four vials were monitoredusing the thermocouples placed inside the liquid contained in each vial.The cryogenic cold gas temperature profile 712 and chamber pressureprofile 714 were then applied to freeze the four vials. The chamberpressure was raised to 30 psig and held at 30 psig for 5 min prior todepressurization. The sharp temperature rise in vial temperatures 716after depressurization indicates that all four vials nucleated and beganfreezing immediately after depressurization. The vial temperatures 716were substantially uniform for the four samples throughout the freezingprocess.

Turning again to FIGS. 12 and 18 b, these figures provide an example ofsample container temperatures prior to and after nucleation of thematerial during a freezing process. The differences in temperaturebetween the samples are greater after nucleation. This change inuniformity of the temperature is due to the latent heat of freezingbeing slightly different for each sample since each sample nucleates ata slightly different temperature. Thus since each sample has a differentlatent heat of freezing and each sample starts at a slightly differenttemperature, the resulting differences in temperature sample-to-sampleare larger after nucleation.

In one embodiment, the uniformity of the container temperatures aremaintained within ±2.5° C. of each other during a freezing process,prior to nucleation, regardless of a location in the cooling chamberwhere the material is frozen. After nucleation, the uniformity of thecontainer temperatures may be maintained within ±5° C. of each otherduring the remainder of the freezing process regardless of a location inthe cooling chamber where the material is frozen. In another embodiment,the uniformity of the container temperatures are maintained within themeasurement error of thermocouple during a freezing process, prior tonucleation, regardless of a location in the cooling chamber where thematerial is frozen. Typical measurement errors are on the order of ±1°C. After nucleation, the uniformity of the container temperatures may bemaintained within ±2.5° C. of each other during the remainder of thefreezing process regardless of a location in the cooling chamber wherethe material is frozen. The above examples of temperature uniformityprior to and after nucleation are non-limiting with respect to thecurrent disclosure.

As seen in FIGS. 12, 15, 16, and 18 b nucleation may be induced usingeither the above described temperature quench or pressure inducednucleation control methods. Nucleation occurs over a smaller time periodas compared to the stochastic nucleation processes presented in FIGS. 13and 14. Furthermore, the above described uniform controlled rate freezermay be adapted to implement either, or both, the temperature quench andpressure induced nucleation methods to selectively induce nucleationsubstantially at the same time in each sample. Therefore, the currentsystems may provide a substantially uniform nucleation time andtemperature for each sample regardless of location within the coolingchamber.

In addition to the above, FIGS. 13 and 14 specifically show thedifferences between a stochastic nucleation process using the currentlydisclosed uniform flow cryogenic chiller and a stochastic nucleationprocess using a prior art system. As illustrated in the figures thesamples nucleated over a smaller temperature range in the uniform flowcryogenic chiller as compared to the prior art system. Without wishingto be bound by theory, this difference in the uniformity of thestochastic nucleation temperatures may be due to the uniform temperatureof the cryogenic cold gas applied to the samples in the uniform flowcryogenic chiller. Therefore, in one embodiment, the currently discloseduniform flow cryogenic chiller may provide a uniform stochasticnucleation temperature to a plurality of containers. In a furtherembodiment, a stochastic nucleation temperature may be provided to10,000, 20,000, 50,000, or 100,000 containers in the same system.

It should be noted that the current invention is not limited to thespecific temperature and pressure profiles described herein. Any numberof variations of the described freezing and nucleation processes will beapparent to one of skill in the art and can be implemented withoutdeparting from the spirit of the current disclosure.

EXAMPLES

The following examples highlight various aspects and features of thepresently disclosed method of inducing nucleation via pressure controlin a material and are not to be taken in a limiting sense. Rather, theseexamples are illustrative only and the scope of the invention should bedetermined only with respect to the claims, appended hereto.

All examples described herein were performed in a pilot-scale VirTis51-SRC freeze-dryer having four shelves with approximately 1.0 squaremeter total shelf space and an internal condenser. This unit wasretrofitted to hold positive pressures of up to about 15 psig. A 1.5″diameter circular opening also was added to the rear wall of thefreeze-drying chamber with 1.5″ diameter stainless steel tubingextending from the hole through the rear wall insulation to emerge fromthe back of the freeze-dryer. Two 1.5″ full-port, air-actuated ballvalves were attached to this tubing via sanitary fittings. One ballvalve allowed gas to flow into the freeze-drying chamber and therebyprovide positive pressures up to 15 psig. The second ball valve allowedgas to flow out of the freeze-drying chamber and thereby reduce chamberpressure to atmospheric conditions (0 psig). All refrigeration of thefreeze-dryer shelves and condenser was accomplished via circulation ofDynalene MV heat transfer fluid cooled by liquid nitrogen using thePraxair NCool™-HX system.

All solutions were prepared in a class 100 clean room. The freeze-dryerwas positioned with the door, shelves, and controls all accessible fromthe clean room while the other components (pumps, heaters, etc.) werelocated in a non-clean room environment. All solutions were preparedwith HPLC grade water (Fisher Scientific, filtered through 0.10 μmmembrane). The final solutions were filtered through a 0.22 μm membraneprior to filling the vials or lyophilization containers. All gases weresupplied via cylinders and were filtered through 0.22 μm filters toremove particulates. The glass containers (5 mL vials and 60 mL bottles)were obtained pre-cleaned for particulates from Wheaton ScienceProducts. Pharmaceutically acceptable carriers were used whereappropriate. The above steps were taken to ensure the materials andmethods met conventional pharmaceutical manufacturing standards forparticulates, which act as nucleating agents.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, antioxidants, salts, coatings,surfactants, preservatives (e.g., methyl or propyl p-hydroxybenzoate,sorbic acid, antibacterial agents, antifungal agents), isotonic agents,solution retarding agents (e.g., paraffin), absorbents (e.g., kaolinclay, bentonite clay), drug stabilizers (e.g., sodium lauryl sulphate),gels, binders (e.g., syrup, acacia, gelatin, sorbitol, tragacanth,polyvinyl pyrrolidone, carboxy-methyl-cellulose, alginates), excipients(e.g., lactose, milk sugar, polyethylene glycol), disintegration agent(e.g., agar-agar, starch, lactose, calcium phosphate, calcium carbonate,alginic acid, sorbitol, glycine), wetting agents (e.g., cetyl alcohol,glycerol monostearate), lubricants, absorption accelerators (e.g.,quaternary ammonium salts), edible oils (e.g., almond oil, coconut oil,oily esters or propylene glycol), sweetening agents, flavoring agents,coloring agents, fillers, (e.g., starch, lactose, sucrose, glucose,mannitol), tabletting lubricants (e.g., magnesium stearate, starch,glucose, lactose, rice flower, chalk), carriers for inhalation (e.g.,hydrocarbon propellants), buffering agents, or such like materials andcombinations thereof, as would be known to one of ordinary skill in theart.

For the experimental conditions described herein and all lyophilizationformulations studied, stochastic nucleation was typically observed tooccur at container temperatures between about −8° C. and −20° C. andoccasionally as warm as −5° C. The containers could generally be held attemperatures warmer than −8° C. for long periods of time withoutnucleating. The onset of nucleation and subsequent crystal growth (i.e.,freezing) was determined by temperature measurement as the point atwhich the container temperature quickly increased in response to theexothermic latent heat of fusion. The initiation of freezing also couldbe visually determined through a sight-glass on the freeze-dryer chamberdoor.

Example 1 Controlling the Nucleation Temperature

Four separate vials were filled with 2.5 mL of 5 wt % mannitol solution.The predicted thermodynamic freezing point of the 5 wt % mannitolsolution is approximately −0.5° C. The four vials were placed on afreeze-dryer shelf in close proximity to one another. The temperaturesof the four vials were monitored using surface mounted thermocouples.The freeze-dryer was pressurized with argon to 14 psig.

The freeze-dryer shelf was cooled to obtain vial temperatures of betweenapproximately −1.3° C. and about −2.3° C. (+1° C. measurement accuracyof the thermocouples). The freeze-dryer was then depressurized fromabout 14 psig to about atmospheric pressure in less than five seconds toinduce nucleation of the solution within the vials. All four vialsnucleated and began freezing immediately after depressurization. Resultsare summarized in Table 1 below.

As seen in Table 1, the controlled nucleation temperatures in thisexample (i.e., Initial Vial Temperatures) are quite close to thepredicted thermodynamic freezing point of the solution. Thus the presentmethod allows control of the nucleation to occur in solutions that havea very low degree of sub-cooling or at nucleation temperatures near oronly slightly colder than their freezing points.

TABLE 1 Controlling the Nucleation Temperature Initial Vial TemperaturePressure Depressurization Vial # Solution Atmos [° C.] Drop [psi]Outcome 1 2.5 mL of 5 wt % mannitol Argon −2.3 14 Nucleation 2 2.5 mL of5 wt % mannitol Argon −1.3 14 Nucleation 3 2.5 mL of 5 wt % mannitolArgon −2.1 14 Nucleation 4 2.5 mL of 5 wt % mannitol Argon −1.7 14Nucleation

Example 2 Controlling the Nucleation Temperature

In this example, ninety-five vials were filled with 2.5 mL of 5 wt %mannitol solution. The thermodynamic freezing point of the 5 wt %mannitol solution is approximately −0.5° C. The ninety-five vials wereplaced on a freeze-dryer shelf in close proximity to one another. Thetemperatures of six vials positioned at different locations in thefreeze-dryer shelf were continuously monitored using surface mountedthermocouples. The freeze-dryer was pressurized in an argon atmosphereto about 14 psig. The freeze-dryer shelf was then cooled to obtain vialtemperatures of near −5° C. The freeze-dryer was then depressurized fromabout 14 psig to about atmospheric pressure in less than five seconds toinduce nucleation of the solution within the vials. All ninety-fivevials were visually observed to nucleate and begin freezing immediatelyafter depressurization. Thermocouple data for the six monitored vialsconfirmed the visual observation. The results are summarized in Table 2.

As seen therein, controlled nucleation temperatures in this example(i.e., Initial Vial Temperatures) are somewhat below the predictedthermodynamic freezing point of the solution. Thus the present methodallows control of the nucleation to occur in solutions that have amoderate degree of sub-cooling. This example also demonstratesscalability of the present method to a multiple vial application.

TABLE 2 Controlling the Nucleation Temperature Initial Vial PressureDepressurization Vial # Solution Atmos Temp [° C.] Drop [psi] Outcome 12.5 mL of 5 wt % mannitol Argon −4.2 14 Nucleation 2 2.5 mL of 5 wt %mannitol Argon −4.4 14 Nucleation 3 2.5 mL of 5 wt % mannitol Argon −4.614 Nucleation 4 2.5 mL of 5 wt % mannitol Argon −4.4 14 Nucleation 5 2.5mL of 5 wt % mannitol Argon −4.6 14 Nucleation 6 2.5 mL of 5 wt %mannitol Argon −5.1 14 Nucleation

Example 3 Controlling the Depressurization Magnitude

In this example, multiple vials were filled with 2.5 mL of 5 wt %mannitol solution. Again, the predicted thermodynamic freezing point ofthe 5 wt % mannitol solution is approximately −0.5° C. For each testrun, the vials were placed on a freeze-dryer shelf in close proximity toone another. As with the earlier described examples, the temperatures ofvials were monitored using surface mounted thermocouples. The argonatmosphere in the freeze-dryer was pressurized to differing pressuresand the freeze-dryer shelf was cooled to obtain vial temperatures ofabout −5° C. In each test run, the freeze-dryer was then rapidlydepressurized (i.e., in less than five seconds) from the selectedpressure to atmospheric pressure in an effort to induce nucleation ofthe solution within the vials. Results are summarized in Table 3.

As seen in Table 3, the controlled nucleation occurred where thepressure drop was about 7 psi or greater and the nucleation temperaturewas between about −4.7° C. and −5.8° C.

TABLE 3 Effect of Depressurization Magnitude Initial Vial PressureDepressurization Vial # Solution Atmos Temp [° C.] Drop [psi] Outcome 12.5 mL of 5 wt % mannitol Argon −4.7 7 Nucleation 2 2.5 mL of 5 wt %mannitol Argon −5.1 7 Nucleation 3 2.5 mL of 5 wt % mannitol Argon −5.37 Nucleation 4 2.5 mL of 5 wt % mannitol Argon −5.6 7 No Nucleation 52.5 mL of 5 wt % mannitol Argon −5.6 7 Nucleation 6 2.5 mL of 5 wt %mannitol Argon −5.8 7 Nucleation 7 2.5 mL of 5 wt % mannitol Argon −5.46 No Nucleation 8 2.5 mL of 5 wt % mannitol Argon −5.7 6 No Nucleation 92.5 mL of 5 wt % mannitol Argon −5.8 6 No Nucleation 10 2.5 mL of 5 wt %mannitol Argon −5.1 5 No Nucleation 11 2.5 mL of 5 wt % mannitol Argon−5.4 5 No Nucleation 12 2.5 mL of 5 wt % mannitol Argon −5.5 5 NoNucleation 13 2.5 mL of 5 wt % mannitol Argon −4.7 4 No Nucleation 142.5 mL of 5 wt % mannitol Argon −5.1 4 No Nucleation 15 2.5 mL of 5 wt %mannitol Argon −5.3 4 No Nucleation

Example 4 Controlling the Depressurization Rates

For this example, multiple vials were filled with about 2.5 mL of 5 wt %mannitol solution having a predicted thermodynamic freezing point ofapproximately −0.5° C. For each test run of varying depressurizationtime, the vials were placed on a freeze-dryer shelf in close proximityto one another. As with the earlier described examples, the temperaturesof vials were monitored using surface mounted thermocouples. Like theabove-described examples, the argon atmosphere in the freeze-dryer waspressurized to about 14 psig and the shelf was cooled to obtain vialtemperatures of approximately −5° C. In each test run, the freeze-dryerwas then depressurized at different depressurization rates from 14 psigto atmospheric pressure in an effort to induce nucleation of thesolution within the vials.

To study the effect of depressurization rate or depressurization time, arestricting ball valve was placed on the outlet of the depressurizationcontrol valve at the rear of the freeze-dryer. When the restrictingvalve is completely open, depressurization from about 14 psig to about 0psig is accomplished in approximately 2.5 seconds. By only partiallyclosing the restricting valve, it is possible to variably increase thechamber depressurization time. Using the restricting ball valve, severaltest runs were performed with the freeze-dryer chamber depressurized atdiffering rates to ascertain or determine the effect of depressurizationrate on nucleation. The results are summarized in Table 4.

TABLE 4 Effect of Depressurization Time Vial Temp Pressure TimeDepressurization Vial # Solution Atmos [° C.] Drop [psi] [sec] Outcome 12.5 mL of 5 wt % mannitol Argon −4.6 14 300 No Nucleation 2 2.5 mL of 5wt % mannitol Argon −5.4 14 300 No Nucleation 3 2.5 mL of 5 wt %mannitol Argon −5.8 14 300 No Nucleation 4 2.5 mL of 5 wt % mannitolArgon −4.6 14 200 No Nucleation 5 2.5 mL of 5 wt % mannitol Argon −5.414 200 No Nucleation 6 2.5 mL of 5 wt % mannitol Argon −5.4 14 200 NoNucleation 7 2.5 mL of 5 wt % mannitol Argon −4.6 14 100 No Nucleation 82.5 mL of 5 wt % mannitol Argon −5.2 14 100 No Nucleation 9 2.5 mL of 5wt % mannitol Argon −5.2 14 100 No Nucleation 10 2.5 mL of 5 wt %mannitol Argon −4.7 14 60 No Nucleation 11 2.5 mL of 5 wt % mannitolArgon −5.1 14 60 No Nucleation 12 2.5 mL of 5 wt % mannitol Argon −5.114 60 No Nucleation 13 2.5 mL of 5 wt % mannitol Argon −5.1 14 50 NoNucleation 14 2.5 mL of 5 wt % mannitol Argon −5.3 14 50 No Nucleation15 2.5 mL of 5 wt % mannitol Argon −4.9 14 50 No Nucleation 16 2.5 mL of5 wt % mannitol Argon −5.4 14 42 No Nucleation 17 2.5 mL of 5 wt %mannitol Argon −5.5 14 42 No Nucleation 18 2.5 mL of 5 wt % mannitolArgon −5.0 14 42 No Nucleation 19 2.5 mL of 5 wt % mannitol Argon −5.114 32 Nucleation 20 2.5 mL of 5 wt % mannitol Argon −5.7 14 32Nucleation 21 2.5 mL of 5 wt % mannitol Argon −5.6 14 32 Nucleation 222.5 mL of 5 wt % mannitol Argon −4.7 14 13 Nucleation 23 2.5 mL of 5 wt% mannitol Argon −5.3 14 13 Nucleation 24 2.5 mL of 5 wt % mannitolArgon −5.5 14 13 Nucleation

As seen in Table 4, nucleation only occurred where the depressurizationtime was less than 42 seconds, the pressure drop was about 14 psi orgreater and the nucleation temperature (i.e., initial vial temperature)was between about −4.6° C. and about −5.8° C. These results indicatethat the depressurization needs to be accomplished relatively quicklyfor the method to be effective.

Example 5 Controlling the Gas Atmosphere

Again, multiple vials were each filled with about 2.5 mL of 5 wt %mannitol solution and placed on a freeze-dryer shelf in close proximityto one another. As with earlier described examples, temperature of thetest vials were monitored using surface mounted thermocouples. For thedifferent test runs, the gas atmosphere in the freeze-dryer was variedalways maintaining a positive pressure of about 14 psig. In thisexample, the freeze-dryer shelf was cooled to obtain vial temperaturesof approximately −5° C. to −7° C. In each test run, the freeze-dryer wasthen rapidly depressurized from about 14 psig to atmospheric pressure inan effort to induce nucleation of the solution within the vials. Theresults are summarized in Table 5.

As seen therein, controlled nucleation occurred in all gas atmospheresexcept for helium gas atmosphere where the pressure drop was about 14psi and the nucleation temperature (i.e., initial vial temperature) wasbetween about −4.7° C. and about −7.4° C. Although not shown in theexamples, it is believed that alternate conditions will likely enablecontrolled nucleation in a helium atmosphere.

TABLE 5 Effect of Gas Atmosphere Composition Initial Vial PressureDepressurization Vial # Solution Atmos Temp [° C.] Drop [psi] Outcome 12.5 mL of 5 wt % mannitol Argon −4.9 14 Nucleation 2 2.5 mL of 5 wt %mannitol Argon −5.2 14 Nucleation 3 2.5 mL of 5 wt % mannitol Nitrogen−4.7 14 Nucleation 4 2.5 mL of 5 wt % mannitol Nitrogen −5.1 14Nucleation 5 2.5 mL of 5 wt % mannitol Xenon −4.8 14 Nucleation 6 2.5 mLof 5 wt % mannitol Xenon −5.0 14 Nucleation 7 2.5 mL of 5 wt % mannitolAir −7.4 14 Nucleation 8 2.5 mL of 5 wt % mannitol Air −7.2 14Nucleation 9 2.5 mL of 5 wt % mannitol Helium −5.8 14 No Nucleation 102.5 mL of 5 wt % mannitol Helium −5.5 14 No Nucleation

Example 6 Large Volume Solutions

In this example, six lyophilization bottles (60 mL capacity) were filledwith about 30 mL of 5 wt % mannitol solution having a predictedthermodynamic freezing point of approximately −0.5° C. The sixlyophilization bottles were placed on a freeze-dryer shelf in closeproximity to one another. The temperature of six bottles positioned atdifferent locations in the freeze-dryer shelf was monitored usingsurface mounted thermocouples. The freeze-dryer was pressurized in anargon atmosphere to about 14 psig. The freeze-dryer shelf was thencooled to obtain bottle temperatures of near −5° C. The freeze-dryer wasthen depressurized from 14 psig to about atmospheric pressure in lessthan five seconds to induce nucleation of the solution within thebottles. The results are summarized in Table 6.

In a separate experiment, a plastic bulk freeze-drying tray (GoreLYOGUARD, 1800 mL capacity) was filled with about 1000 mL of 5 wt %mannitol solution. The tray was obtained pre-cleaned to meet USP lowparticulate requirements. The tray was placed on a freeze-dryer shelf,and the temperature of the tray was monitored by a thermocouple mountedon the exterior surface of the tray near the center of one side. Thefreeze-dryer shelf was then cooled to obtain a tray temperature of near−7° C. The freeze-dryer was then depressurized from 14 psig to aboutatmospheric pressure in less than five seconds to induce nucleation ofthe solution within the tray. The results are also summarized in Table6.

Like the above-described examples, all containers nucleated and beganfreezing immediately after depressurization. Also like theabove-described examples, the nucleation temperatures (i.e., ContainerTemperatures) in this example were very much controllable to be somewhatnear the thermodynamic freezing temperature of the solution. Moreimportantly, this example illustrates that the present method allowscontrol of the nucleation to occur in larger volume solutions andvarious container formats. It should be noted that one would expect theefficacy of the depressurization method to improve as formulation volumeincreases, because the nucleation event is more likely to occur whenmore molecules are present to aggregate and form critical nuclei.

TABLE 6 Effect of Solution Volume and Container Type ContainerTemperature Pressure Depressurization Container Solution Atmos [° C.]Drop [psi] Outcome Bottle # 1 30 mL of 5 wt % mannitol Argon −5.3 14Nucleation Bottle # 2 30 mL of 5 wt % mannitol Argon −5.1 14 NucleationBottle # 3 30 mL of 5 wt % mannitol Argon −5.9 14 Nucleation Bottle # 430 mL of 5 wt % mannitol Argon −5.2 14 Nucleation Bottle # 5 30 mL of 5wt % mannitol Argon −5.9 14 Nucleation Bottle # 6 30 mL of 5 wt %mannitol Argon −6.1 14 Nucleation Tray 1000 mL of 5 wt % mannitol Argon−6.9 14 Nucleation

Example 7 Dynamic Cooling vs. Equilibrated Cooling

The present methods of controlling nucleation can be used in variousmodes. Examples 1-6, described above, each demonstrate the aspect ofcontrolling the nucleation temperature of a lyophilization solution thatis essentially equilibrated at a temperature below its thermodynamicfreezing point (i.e., very slowly changing temperature). This exampledemonstrates that nucleation can also occur at a temperature below thethermodynamic freezing point in a dynamic cooling environment (i.e., thesolution is undergoing rapid changes in temperature).

In this example, vials 1 through 6 represent the samples described abovewith reference to Example 2. In addition, three separate vials (Vials7-9) were also filled with 2.5 mL of 5 wt % mannitol solution. In aseparate test run, the three additional vials were placed on afreeze-dryer shelf in close proximity to one another. The freeze-dryershelf was cooled rapidly towards a final shelf temperature of −45° C.When one of the vials reached a temperature of about −5° C., as measuredby the surface mounted thermocouples, the freeze-dryer was depressurizedrapidly from about 14 psig to 0 psig in an effort to induce nucleation.All three vials nucleated and began freezing immediately afterdepressurization. The vial temperatures decreased significantly tobetween −6.8° C. and −9.9° C. prior to nucleation as a result of thedynamic cooling environment. Comparative results are summarized in Table7 below.

TABLE 7 Test Results - Effect of Dynamic Cooling on NucleationNucleation Pressure Depressurization Vial # Solution Mode Temp. [° C.]Drop [psi] Outcome 1 2.5 mL of 5 wt % mannitol Equilibrated −4.2 14Nucleation 2 2.5 mL of 5 wt % mannitol Equilibrated −4.4 14 Nucleation 32.5 mL of 5 wt % mannitol Equilibrated −4.6 14 Nucleation 4 2.5 mL of 5wt % mannitol Equilibrated −4.4 14 Nucleation 5 2.5 mL of 5 wt %mannitol Equilibrated −4.6 14 Nucleation 6 2.5 mL of 5 wt % mannitolEquilibrated −5.1 14 Nucleation 7 2.5 mL of 5 wt % mannitol Dynamic −6.814 Nucleation 8 2.5 mL of 5 wt % mannitol Dynamic −7.2 14 Nucleation 92.5 mL of 5 wt % mannitol Dynamic −9.9 14 Nucleation

The efficacy of the present methods for controlling nucleation inlyophilization solutions equilibrated in a given temperature range orlyophilization solutions being dynamically cooled, provides the end-userwith two potential modes of application with different benefits andtrade-offs. By allowing the lyophilization solutions to equilibrate, therange of nucleation temperatures will be narrow or minimized to theperformance limits of the freeze-dryer itself. The equilibration stepmay require extra time to achieve relative to conventional or dynamicfreezing protocols where the chamber and vial temperatures are droppedto less than about −40° C. in one step. However, employing theequilibration step should yield much improved nucleation uniformityacross all vials or containers as well as realization of the otherbenefits associated with precisely controlling the nucleationtemperature of the material.

Alternatively, if equilibrating the material or lyophilization solutiontemperatures is undesirable, one may simply implement thedepressurization step at an appropriate time during the normal freezingor dynamic cooling protocol. Depressurization during a dynamic cool downwill produce a wider spread in nucleation temperatures for the materialwithin the lyophilization containers, but will add minimal time to thefreezing protocol and still allow one to mitigate the problems ofextreme sub-cooling.

Example 8 Effect of Different Excipients

The present method of controlling or inducing nucleation in a materialcan be used to control the nucleation temperature of sub-cooledsolutions containing different lyophilization excipients. This exampledemonstrates the use of the present methods with the followingexcipients: mannitol; hydroxyethyl starch (HES); polyethylene glycol(PEG); polyvinyl pyrrolidone (PVP); dextran; glycine; sorbitol; sucrose;and trehalose. For each excipient, two vials were filled with 2.5 mL ofa solution containing 5 wt % of the excipient. The vials were placed ona freeze-dryer shelf in close proximity to one another. The freeze-dryerwas pressurized in an argon atmosphere to about 14 psig. Thefreeze-dryer shelf was cooled to obtain vial temperatures near −3° C.and then depressurized rapidly to induce nucleation. Results aresummarized in Table 8.

TABLE 8 Effect of Different Lyophilization Excipients Initial VialTemperature Pressure Depressurization Vial # Solution/Excipient Atmos [°C.] Drop [psi] Outcome 1 2.5 mL of 5 wt % mannitol Argon −3.3 14Nucleation 2 2.5 mL of 5 wt % mannitol Argon −3.0 14 Nucleation 3 2.5 mLof 5 wt % HES Argon −3.1 14 Nucleation 4 2.5 mL of 5 wt % HES Argon −3.714 Nucleation 5 2.5 mL of 5 wt % PEG Argon −3.8 14 Nucleation 6 2.5 mLof 5 wt % PEG Argon −3.4 14 Nucleation 7 2.5 mL of 5 wt % PVP Argon −3.514 Nucleation 8 2.5 mL of 5 wt % PVP Argon −3.3 14 Nucleation 9 2.5 mLof 5 wt % dextran Argon −4.0 14 Nucleation 10 2.5 mL of 5 wt % dextranArgon −3.1 14 Nucleation 11 2.5 mL of 5 wt % glycine Argon −3.8 14Nucleation 12 2.5 mL of 5 wt % glycine Argon −3.9 14 Nucleation 13 2.5mL of 5 wt % sorbitol Argon −3.6 14 Nucleation 14 2.5 mL of 5 wt %sorbitol Argon −3.4 14 Nucleation 15 2.5 mL of 5 wt % sucrose Argon −3.314 Nucleation 16 2.5 mL of 5 wt % sucrose Argon −3.4 14 Nucleation 172.5 mL of 5 wt % trehalose Argon −3.7 14 Nucleation 18 2.5 mL of 5 wt %trehalose Argon −3.1 14 Nucleation

Example 9 Controlling Nucleation of Protein Solutions

The methods disclosed herein can be used to control the nucleationtemperature of sub-cooled protein solutions without negative or adverseeffects on protein solubility or enzymatic activity. Two proteins,bovine serum albumin (BSA) and lactate dehydrogenase (LDH) were used inthis example.

BSA was dissolved in 5 wt % mannitol at a concentration of 10 mg/mL.Three lyophilization vials were filled with 2.5 mL of the BSA-mannitolsolution and placed on a freeze-dryer shelf in close proximity to oneanother. The freeze-dryer was pressurized in an argon atmosphere toabout 14 psig. The freeze-dryer shelf was cooled to obtain vialtemperatures near −5° C. The freeze-dryer was depressurized rapidly toinduce nucleation. All vials of BSA solution nucleated and beganfreezing immediately after depressurization. No precipitation of theprotein was observed upon thawing.

The LDH proteins were obtained from two different suppliers and forpurposes of clarity are designated as LDH-1 or LDH-2 to distinguish thetwo distinct batches. LDH-1 was dissolved in 5 wt % mannitol at aconcentration of 1 mg/mL. Six lyophilization vials were filled with 2.5mL of the LDH-1/mannitol solution and placed on a freeze-dryer shelf inclose proximity to one another. The freeze-dryer was pressurized in anargon atmosphere to about 14 psig. The freeze-dryer shelf was cooledstarting from room temperature to obtain vial temperatures near −4° C.The freeze-dryer was then depressurized rapidly to induce nucleation.All vials nucleated and began freezing immediately afterdepressurization. The vials were held at this state for about 15minutes. The freeze-dryer shelf was then cooled at a rate ofapproximately 1° C./min to obtain vial temperatures near −45° C. andheld for an additional 15 minutes to ensure completion of the freezingprocess. After the freezing step, the freeze-dryer shelf was then warmedat a rate of about 1° C./min to raise the vial temperatures to near 5°C. No precipitation of the protein was observed upon thawing. The vialcontents were assayed for enzymatic activity, and the results werecompared to a control sample of unfrozen LDH-1/mannitol solution.

As part of Example 9, the depressurized nucleated samples of theLDH-1/mannitol solution were compared to stochastically nucleatedsamples. In the stochastically nucleated samples of LDH-1, the procedurewas repeated without pressurization and depressurization and without theargon atmosphere. Specifically, LDH-1 was dissolved in 5 wt % mannitolat a concentration of 1 mg/mL. Six lyophilization vials were filled with2.5 mL of the LDH-1/mannitol solution and placed on a freeze-dryer shelfin close proximity to one another. The freeze-dryer shelf was cooledstarting from room temperature at a rate of about 1° C./min to obtainvial temperatures near −45° C. and held for 15 minutes to ensurecompletion of the freezing process. After the freezing step, thefreeze-dryer shelf was warmed at a rate of about 1° C./min to raise thevial temperatures to near 5° C. No precipitation of the protein wasobserved upon thawing. The vial contents were assayed for enzymaticactivity, and the results were compared to the same control sample ofunfrozen LDH-1/mannitol solution. Also as part of Example 9, theexperiments described above for LDH-1 were repeated using LDH-2. Theonly difference was a nucleation temperature near −3° C. for LDH-2rather than −4° C. for LDH-1.

As seen in Table 9, the controlled nucleation and freezing processachieved via depressurization clearly does not decrease enzymaticactivity relative to a comparable stochastic nucleation and freezingprotocol. In fact, the controlled nucleation process achieved viadepressurization appears to better preserve enzyme activity with a meanactivity loss of only 17.8% for LDH-1 and 26.5% for LDH-2 compared tothe mean activity loss of 35.9% for LDH-1 and 41.3% for LDH-2 afterstochastic nucleation.

TABLE 9 Controlling the Nucleation Temperature of Sub-Cooled ProteinSolutions Vial Temp Pressure Enzyme Activity Depressurization Vial #Solution Atmos [° C.] Drop[psi] Loss [%] Outcome 1 2.5 mL of BSAsolution Argon −4.9 14 — Nucleation 2 2.5 mL of BSA solution Argon −4.314 — Nucleation 3 2.5 mL of BSA solution Argon −5.3 14 — Nucleation 42.5 mL of LDH-1 solution Argon −3.8 14 9.0 Nucleation 5 2.5 mL of LDH-1solution Argon −4.0 14 16.2 Nucleation 6 2.5 mL of LDH-1 solution Argon−3.7 14 18.4 Nucleation 7 2.5 mL of LDH-1 solution Argon −4.0 14 23.4Nucleation 8 2.5 mL of LDH-1 solution Argon −3.9 14 18.5 Nucleation 92.5 mL of LDH-1 solution Argon −4.0 14 21.2 Nucleation 10 2.5 mL ofLDH-1 solution Air −10.4 0 35.7 Nucleation 11 2.5 mL of LDH-1 solutionAir −16.5 0 35.4 Nucleation 12 2.5 mL of LDH-1 solution Air −15.5 0 36.1Nucleation 13 2.5 mL of LDH-1 solution Air −10.5 0 43.9 Nucleation 142.5 mL of LDH-1 solution Air −9.8 0 24.9 Nucleation 15 2.5 mL of LDH-1solution Air −11.0 0 39.2 Nucleation 16 2.5 mL of LDH-2 solution Argon−3.1 14 29.9 Nucleation 17 2.5 mL of LDH-2 solution Argon −2.9 14 18.9Nucleation 18 2.5 mL of LDH-2 solution Argon −3.1 14 23.3 Nucleation 192.5 mL of LDH-2 solution Argon −2.7 14 19.6 Nucleation 20 2.5 mL ofLDH-2 solution Argon −3.1 14 32.1 Nucleation 21 2.5 mL of LDH-2 solutionArgon −2.6 14 35.2 Nucleation 22 2.5 mL of LDH-2 solution Air −5.0 038.3 Nucleation 23 2.5 mL of LDH-2 solution Air −5.5 0 40.0 Nucleation24 2.5 mL of LDH-2 solution Air −2.3 0 36.5 Nucleation 25 2.5 mL ofLDH-2 solution Air −3.8 0 42.0 Nucleation 26 2.5 mL of LDH-2 solutionAir −5.1 0 50.2 Nucleation 27 2.5 mL of LDH-2 solution Air −5.9 0 40.6Nucleation

It should be noted that the stochastic nucleation temperatures observedfor LDH-2 were substantially warmer than the stochastic nucleationtemperatures for LDH-1. This difference may be due to some contaminantacting as a nucleating agent in the LDH-2. The stochastic nucleationtemperatures are much closer to the controlled nucleation temperaturesfor LDH-2 compared to LDH-1, yet the improvements in retention of enzymeactivity obtained via controlled nucleation for LDH-1 and LDH-2 aresimilar at 18.1% and 14.8%, respectively. This result suggests that theimprovements in retention of enzyme activity can be partially attributedto the characteristics of the controlled nucleation process itself, notjust to the prescribed warmer nucleation temperatures obtained viadepressurization.

Example 10 Reducing Primary Drying Time

A 5 wt % mannitol solution was prepared by mixing about 10.01 grams ofmannitol with about 190.07 grams of water. Vials were filled with 2.5 mLof the 5 wt % mannitol solution. The vials were weighed empty and withthe solution to determine the mass of water added to the vials. Thetwenty vials were placed in a rack on a freeze-dryer shelf in closeproximity to one another. The temperatures of six vials were monitoredusing surface mounted thermocouples; all monitored vials were surroundedby other vials to improve uniformity of vial behavior. The freeze-dryerwas pressurized to about 14 psig in a controlled gas atmosphere of argongas. The freeze-dryer shelf was cooled from room temperature to about−6° C. to obtain vial temperatures of between approximately −1° C. and−2° C. The freeze-dryer was then depressurized from about 14 psig toabout atmospheric pressure in less than five seconds to inducenucleation of the solution within the vials. All vials observed visuallyor monitored via thermocouples nucleated and began freezing immediatelyafter depressurization. The shelf temperature was then lowered rapidlyto about −45° C. to complete the freezing process. Once all vialtemperatures were about −40° C. or less, the freeze-drying chamber wasevacuated and the process of primary drying (i.e., sublimation) wasinitiated. During this drying process, the freeze-dryer shelf was warmedto about −14° C. via a one hour ramp and held at that temperature for 16hours. The condenser was maintained at about −60° C. throughout thedrying process. Primary drying was stopped by turning off the vacuumpump and backfilling the chamber with argon to atmospheric pressure. Thevials were promptly removed from the freeze-dryer and weighed todetermine how much water was lost during the primary drying process.

In a separate experiment as part of Example 10, other vials were filledwith 2.5 mL of the same 5 wt % mannitol solution. The vials were weighedempty and with the solution to determine the mass of water added to thevials. The vials were loaded into the freeze-dryer in the same mannerdescribed above, and the temperatures of six vials were once againmonitored using surface-mounted thermocouples. The freeze-dryer shelfwas cooled rapidly from room temperature to about −45° C. to freeze thevials. Nucleation occurred stochastically between about −15° C. andabout −18° C. during the cooling step. Once all vials temperatures wereabout −40° C. or less, the vials were dried in a manner identical to themethod described above. Upon conclusion of primary drying, the sampleswere promptly removed from the freeze-dryer and weighed to determine howmuch water was lost during the primary drying process.

TABLE 10 Increasing the Nucleation Temperature Improves Primary DryingInitial Vial Water Temp. Pressure Loss Depressurization Vial # SolutionAtmos [° C.] Drop [psi] [%] Outcome 1 2.5 mL of 5 wt % mannitol Argon−1.3 14 89.9 Nucleation 2 2.5 mL of 5 wt % mannitol Argon −1.9 14 85.2Nucleation 3 2.5 mL of 5 wt % mannitol Argon −1.3 14 87.1 Nucleation 42.5 mL of 5 wt % mannitol Argon −2.3 14 88.8 Nucleation 5 2.5 mL of 5 wt% mannitol Argon −2.1 14 85.0 Nucleation 6 2.5 mL of 5 wt % mannitolArgon −1.1 14 80.7 Nucleation 7 2.5 mL of 5 wt % mannitol Air −15.7 065.7 — 8 2.5 mL of 5 wt % mannitol Air −16.7 0 66.9 — 9 2.5 mL of 5 wt %mannitol Air −14.5 0 64.6 — 10 2.5 mL of 5 wt % mannitol Air −15.6 064.7 — 11 2.5 mL of 5 wt % mannitol Air −16.5 0 64.1 — 12 2.5 mL of 5 wt% mannitol Air −17.9 0 65.7 —

Results of the freeze-drying process with controlled nucleation andstochastic nucleation are summarized in Table 10. It should be notedthat these two experiments only differ in the addition of the controllednucleation via depressurization step to one experiment. As seen in Table10, the controlled nucleation process achieved via depressurizationallows nucleation at very low degrees of sub-cooling, between about−1.1° C. and −2.3° C. in this example. The much warmer nucleationtemperatures for the controlled nucleation case compared to thestochastic nucleation case yields an ice structure and resultantlyophilized cake with dramatically improved drying properties. For thesame amount of drying time, the vials nucleated using the discloseddepressurization methods between about −1.1° C. and −2.3° C. lost anaverage of 86.1% of their water while the vials nucleated stochasticallybetween about −14.5° C. and −17.9° C. only lost an average of 65.3%.Hence, the vials nucleated stochastically would require much moreprimary drying time to achieve the same degree of water loss as thevials nucleated in a controlled manner in accordance with the presentlydisclosed methods. The improvement in drying time is likely attributedto the formation of larger ice crystals at warmer nucleationtemperatures. These larger ice crystals leave behind larger pores uponsublimation, and the larger pores offer less resistance to the flow ofwater vapor during further sublimation.

Another benefit associated with the above presented temperature quenchand pressure induced nucleation control methods is that by controllingthe lowest nucleation temperature and/or the precise time of nucleationone can affect the ice crystal structure formed within the frozen vialsor containers. The ice crystal structure is a variable that affectsvarious properties of the preserved material, including but not limitedto, activity, functionality, and viability as well as the time it takesfor ice to sublimate during a freeze-drying process. Thus, controllingthe ice crystal structure is important for both cryopreservation andfreeze-drying processes.

Enhanced Viability and Biological Activity

As detailed above, FIG. 11 is an illustrative temperature profile of thecryogenic cold gas used to cool samples in the presently discloseduniform flow controlled rate freezer. The temperature profile includesan equilibrium step 402, a cooling step 404, a pre-nucleationtemperature step 406, a temperature quench step 408, a temperaturequench hold step 410, a post-nucleation temperature hold step 412, and afinal cooling step 414. The testing presented below was done to optimizethe presented temperature profile to maximize the cell viability duringa cryopreservation process utilizing a temperature quench method toinduce nucleation. While results are presented for a temperature quenchcontrolled nucleation, a pressure controlled nucleation process could beused to obtain similar results with regards to cell viability.

Furthermore, while viability results are presented for living cells, itwould be apparent to one of skill in the art that the described systemsand methods could be used to provide a uniform biological activity fornon-living materials.

Cell cultures of Normal Human Dermal Fibroblasts (NHDF) were obtainedfrom Lonza (Walkersville, Md.). Stock cultures were maintained at 37° C.in 95% air/5% CO₂ in Falcon T-75 cm²-flasks. NHDF cultures were grown inFibroblast Basal Medium (FBM) supplemented with Fibroblast Growth Medium(FGM SingleQuots supplied by Lonza). Stock cultures were subculturedevery 5-6 days at approximately 95% confluence, and media wasreplenished every 3 days. Experiments were performed using cell culturesbetween passages 2 and 10. One day prior to experimentation, cultureswere supplemented with fresh culture media.

In-house media/Dimethyl sulfoxide (DMSO) solutions were prepared by theaddition of DMSO (5% v/v) to the complete FGM (having 10% fetal bovineserum). For testing purposes the solution containing in-house media with5% v/v DMSO is labeled as “Media+5% DMSO”. The intracellular-likeCryoStor solution (BioLife Solutions, WA) is serum-free and protein-freeand is premixed with 5% v/v DMSO. For testing purposes the solutioncontaining CryoStor solution premixed with 5% v/v DMSO is labeled as“CS5”.

To test solutions for cryopreservation efficacy, standardcryopreservation methods were performed. Briefly, cells (1×10⁶ cells/ml)were re-suspended in 0.5 ml of the respective solutions to be tested andplaced into 1.2 ml cryovials. Cryopreservation studies were performedusing the uniform controlled rate freezer currently disclosed. Sampleswere stored for 10 minutes at 2-8° C. in the freezing chamber to alloweach of the samples to equilibrate in temperature prior to beingsubjected to various freezing protocols. Following the cryogenicfreezing process, samples were immediately transferred into liquidnitrogen for 18-24 hours. Samples were thawed in a 37° C. water bath,immediately re-suspended in culture media (1:10 dilution), plated in awell sample plate, and allowed to recover for one day prior toassessment.

Cell viability was assessed for each freezing process both qualitativelyand quantitatively. Qualitative assessment was achieved by visualizationusing light microscopy. Quantitative assessment was conducted usingAlamarBlue™ (AbD Serotec) for florescence spectroscopy evaluation.AlamarBlue™ was diluted in a ratio of 1:20 in Hank's Balanced SaltSolution without phenol red (HBSS) available from Life Technologies,Gaithersburg, Md. 100 μl of the culture medium was removed from eachwell in the well sample plates and 100 μl of the working AlamarBlue™solution was added to each well for analysis. Samples were subsequentlyincubated in the dark at 37° C. for 60 min (+1 min) The fluorescence ofeach sample was then evaluated using a Tecan SPECTRAFluor Plus platereader (TECAN Austria GmbH) with a 530-nm excitation/590-nm emissionfilter set. This assessment was performed 24 hours post-preservation foreach experiment.

The above disclosed testing methods were used to optimize the coolingrates, hold times, and temperatures of the cryogenic cold gastemperature profile with respect to the cell viability of NHDF cellsduring a temperature quench controlled nucleation freezing process. Theresults of the testing are presented in FIGS. 11, 12-13, and 19-31. Thetesting results in FIGS. 19-31 correspond to the cryogenic cold gastemperature profile steps defined in FIG. 11.

The samples presented in FIGS. 12 and 13 were prepared using CS5solution and the above disclosed method. Testing was conducted using thecurrently disclosed uniform flow cryogenic chiller unit. As detailedabove, FIG. 12 depicts temperature profiles of samples that haveundergone a freezing process with temperature quench nucleation control.FIG. 13 depicts temperature profiles of samples that have undergone afreezing process with no nucleation control and a constant cooling rateof 5° C./min. Tables 11 and 12 present measured nucleation temperatureand percent viability of the samples depicted in FIGS. 12 and 13 forsamples with temperature quench nucleation control and withoutnucleation control, respectively.

TABLE 11 Nucleation Temperature and Viability of Samples withTemperature Quench Nucleation Control Nucleation Vial # Temperature (°C.) % Viability Vial 1 7.2 80 Vial 2 8.1 84 Vial 3 8.0 75 Vial 4 7.0 78Vial 5 7.5 79 Vial 6 7.5 78

TABLE 12 Nucleation Temperature and Viability of Samples withoutNucleation Control Nucleation Vial # Temperature (° C.) % Viability Vial1 11.8 62 Vial 2 15.2 64 Vial 3 10.2 68 Vial 4 9.3 65 Vial 5 10.3 68Vial 6 11.9 65

Table 13 presents additional data from testing of NHDF cells preparedusing CS5 solution and the above detailed method and the currentlydisclosed uniform flow cryogenic chiller unit. Table 13 presents datafor samples frozen without nucleation control at different constantcooling rates. Optimal NHDF cell recovery, for a process withoutnucleation control, was observed for cooling rates of 1° C./min, 5°C./min, and 10° C./min Decreased cell recovery was observed with aslower cooling rate of 0.5° C./min. Decreased cell recovery was alsoobserved for faster cooling rates of 15 and 25° C./min.

TABLE 13 Viability of Samples without Nucleation Control and DifferentCooling Rates Cooling Rate (° C./min) % Viability 0.5 58 1 69 5 67 10 6815 60 25 41

As expected the samples with nucleation control, presented in Table 11,exhibited a smaller variance in the observed nucleation temperature ascompared to the samples without nucleation, presented in Table 12.Furthermore, the samples that underwent the nucleation control exhibitan enhanced viability as compared to the data presented in Tables 12 and13. Specifically, the average viability from the temperature quenchinduced nucleation samples is 79% as compared to the range ofviabilities shown in Tables 12 and 13 ranging from 41% to 69% withoutnucleation control. As stated above testing was conducted with the abovedisclosed uniform flow cryogenic chiller unit. Therefore, if the abovefreezing process with nucleation control were to be scaled up using theuniform flow cryogenic chiller unit there will be minimal sample tosample variation in temperature profile between the plurality of samplesfor any number of samples. Such a process would result in the pluralityof samples having a uniform enhanced viability with minimal differencesfrom vial to vial and batch to batch for any number of samples. In acertain embodiments such a system could include at least 10,000, 20,000,50,000, or 100,000 samples.

FIG. 19 illustrates the effect of Pre-nucleation temperature on the cellviability of NHDF cells following cryopreservation. Curve 800corresponds to the tests conducted with CS5 solution and curve 802corresponds to the tests conducted with Media+5% DMSO solution. For thistest, the pre-nucleation temperature was set at −2.5° C., −5° C., −7.5°C. and 10° C. respectively while the remaining cooling parameters werekept the same. Optimal NHDF cell viability in both solutions wasobserved for a pre-nucleation temperature of −5° C. Decreased cellviability was observed for warmer and colder pre-nucleationtemperatures.

FIG. 20 shows the effect of the quench temperature on the cell viabilityof NHDF cells following cryopreservation. Curve 804 corresponds to thetests conducted with CS5 solution and curve 806 corresponds to the testsconducted with Media+5% DMSO solution. After being held at thepre-nucleation temperature, the cryogenic cold gas was rapidly decreasedto −40° C., −60° C., −70° C. and −80° C. to initiate nucleation. Afterperforming the temperature quench, the cryogenic cold gas temperaturewas raised to −20° C. at a rate of 30° C./min and held at −20° C. for 10minutes. The cryogenic cold gas was then cooled to −80° C. at a rate of−2.5° C./min Optimal NHDF cell viability in both solutions was observedfor a quench temperature of −80° C. NHDF cell viability slightlydecreased for quench temperatures of −60° C. and −70° C., while asignificant decrease was observed for a quench temperature of −40° C.

FIG. 21 compares the sample temperature profiles for the tests depictedin FIG. 20 with different quench temperatures for nucleation control forthose samples tested in CS5 solution. The tested quench temperatureswere −40° C. (curve 808), −60° C. (curve 810), −70° C. (curve 812), and−80° C. (curve 814). Combined with FIG. 20, it was observed thatnucleation temperature and the sample post-nucleation cooling rate wererelated to the quench temperature. Furthermore, the combination of thenucleation temperature and post-nucleation cooling rate affect cellrecovery. The moderate sample cooling rate observed for a quenchtemperature of about −80° C. gave the optimal recovery.

FIG. 22 illustrates the effect of the quench temperature and the holdtime of the quench on the viability of NHDF cells followingcryopreservation. Curve 816 corresponds to the tests conducted with CS5solution and curve 818 corresponds to the tests conducted with Media+5%DMSO solution. After being held at the pre-nucleation temperature, thecryogenic cold gas temperature was rapidly decreased to −80° C. with nohold time, −80° C. with a two minute hold, and −100° C. with aone-minute hold. After performing the respective temperature quenchesand hold, the cryogenic cold gas was warmed up to −35° C. at 30° C./minand held for 10 minutes. The cryogenic cold gas was then cooled down to−80° C. at 2.5° C./min. The different temperatures and hold times wereevaluated to determine the effect of latent heat on cell viability.Looking at FIG. 22, applying colder quench temperatures (e.g. −100° C.)or increasing the holding time at the quench temperature to minimizelatent heat resulted in decreased cell viability. Optimal NHDF cellviability in both solutions was observed for a quench temperature of−80° C. with no quench holding time.

FIG. 23 shows the temperature profiles associated with the testsdepicted in FIG. 22 with different quench temperatures and hold timesfor nucleation control for those samples tested in CS5 solution. Theprofiles parameters were −80° C. with no hold (curve 820), −80° C. witha 2 minute hold (curve 822), and −100° C. with a one minute hold (curve824).

FIG. 24 presents the effect of the post-nucleation hold temperature onthe viability of NHDF cells following cryopreservation. Curve 826corresponds to the tests conducted with CS5 solution and curve 828corresponds to the tests conducted with Media+5% DMSO solution. Afterbeing held at the pre-nucleation temperature, the cryogenic cold gas wasrapidly decreased to −80° C. with no hold time, then the cryogenic coldgas was warmed up to a post-nucleation holding temperature of −10° C.,−20° C., −35° C. or −50° C. at a rate of 30° C./min and held for 10minutes prior to further cooling to −80° C. at a rate of 2.5° C./min Nosignificant differences were observed in the post thaw viability of NHDFcells for post-nucleation holding temperatures of −10° C., −20° C., −35°C. or −50° C. for the CS5 media solution. A decreased viability of NHDFcells was observed at a post-nucleation holding temperature of −10° C.for the media+5% DMSO solution.

FIG. 25 shows the temperature profiles associated with the testsdepicted in FIG. 24 with different post-nucleation holding temperaturesfor those samples tested in CS5 solution. The different post-nucleationhold temperatures were −10° C. (curve 830), −20° C. (curve 832), −35° C.(curve 834), and −50° C. (curve 836).

FIG. 26 shows the effect of post-nucleation holding time on theviability of NHDF cells following cryopreservation. Curve 838corresponds to the tests conducted with CS5 solution and curve 840corresponds to the tests conducted with Media+5% DMSO solution. Afterreaching the pre-nucleation temperature, the cryogenic cold gas wasplunged to −80° C. with no hold time. The cryogenic cold gas was thenwarmed up to a post nucleation temperature of −35° C. at a rate of 30°C./min and held at −35° C. for different times ranging from no hold timeto twenty minutes prior to further cooling to −80° C. at a rate of 2.5°C./min. The test run without a post-nucleation hold time resulted in thelowest cell viability. There was a roughly 10%-20% difference inviability observed between the tests with various hold times at apost-nucleation hold temperature of −35° C. for CS5 media solution andmedia+5% DMSO. Optimal cell viability was observed in both solutions ata post-nucleation hold temperature of −35° C. with a 10 minutepost-nucleation holding time.

FIG. 27 shows the temperature profiles associated with the testsdepicted in FIG. 26 with different post-nucleation hold times at −35° C.for those samples tested in CS5 solution. The different post-nucleationhold times were no hold (curve 842), 2 minutes (curve 844), 5 minutes(curve 846), 10 minutes (curve 848), and 20 minutes (curve 850).

FIGS. 28 and 30 illustrate the effect of the post-nucleation coolingrate following the post-nucleation temperature hold on the viability ofNHDF cells following cryopreservation. FIG. 28 was conducted with a −35°C. post-nucleation hold temperature. FIG. 30 was conducted with a −10°C. post-nucleation hold temperature. Curves 852 and 862 correspond tothe tests conducted with CS5 solution and curves 854 and 864 correspondto the tests conducted with Media+5% DMSO solution. After being held atthe pre-nucleation temperature, the cryogenic cold gas was rapidlydecreased to −80° C. with no hold time, The cryogenic cold gas was thenwarmed up to a post nucleation temperature of −35° C. or −10° C. at arate of 30° C./min and held at the post-nucleation temperature for 10minutes. The samples were then further cooled to −80° C. at differentrates ranging from 2.5 to 150° C./min. It was observed that the warmerpost-nucleation temperature of −10° C. resulted in increased sensitivityof the cell viability to the applied cooling rate. The difference incell viability between a cooling rate of 10° C./min and 150° C./min wasapproximately 5% for the post-nucleation temperature of −35° C. andapproximately 10% for the post-nucleation temperature of −10° C.

FIG. 29 shows the temperature profiles associated with the postnucleation cooling rates shown in FIG. 28 and tested with a −35° C.post-nucleation hold temperature in CS5 solution. The differentpost-nucleation cooling rates were 2.5° C./min (curve 856), 10° C./min(curve 858), and 150° C./min (curve 860).

FIG. 31 shows the temperature profiles associated with the postnucleation cooling rates shown in FIG. 30 and tested with a −10° C.post-nucleation hold temperature in CS5 solution.

The different post-nucleation cooling rates were 2.5° C./min (curve866), 10° C./min (curve 868), 20° C./min (curve 870), and 150° C./min(curve 872).

In combination, the above disclosed cooling and nucleation methods andthe uniform flow controlled rate freezer are capable of providinguniformly enhanced viability of biological material frozen in any numberof vials or containers located within a single system. One such systemmight be a uniform flow controlled rate freezer incorporating multipleuniform flow cryogenic chiller unit modules. Such a system would becapable of providing uniformly enhanced viability to at least 10,000,20,000, 50,000, or 100,000 vials or containers containing biologicalmaterial.

Alternatively, the uniform flow controlled rate freezer could be used toimplement simpler cooling profiles without nucleation control such asthe cooling rate presented in FIG. 13. Such a method would result insimilar results for the viability of the frozen material as provided byprior art methods and systems. However, as detailed above, in contrastto prior art systems the uniform flow controlled rate freezer is capableof providing a uniform temperature and flow of cryogenic cold gas toeach sample which will result in a uniform viability for each sampleregardless of location within the system or cooling chamber in which theplurality of containers were frozen. Therefore, even when only applyinga simple cooling profile without nucleation control, the currentlydisclosed devices and methods are capable of providing a uniformviability of biological material frozen in any number of vials orcontainers located within a single system. In a first embodiment thenumber of samples exhibiting a uniform viability is at least 50,000. Ina second embodiment the number of samples exhibiting a uniform viabilityis at least 100,000.

The present method provides an improved method for controlling thetemperature and/or time at which sub-cooled materials, namely liquids orsolutions, nucleate and then freeze. Although this application focusesin part on freeze-drying, a similar problem occurs for any materialprocessing step that involves a nucleated phase transition. Examples ofsuch processes include the crystallization of polymers and metals frommelts, crystallization of materials from supersaturated solutions,crystallization of proteins, artificial snow production, food freezing,freeze concentration, fractional crystallization, cryo-preservation, orcondensation of vapors to liquids.

The most immediate benefit of controlling the nucleation temperature ofa liquid or solution is the ability to control the number and size ofthe solid domains produced by the phase transition. In freezing water,for example, the nucleation temperature directly controls the size andnumber of ice crystals formed. Generally speaking, the ice crystals arefewer in number and larger in size when the nucleation temperature iswarmer.

The ability to control the number and size of the solid domains producedby a phase transition may provide additional benefits. In afreeze-drying process, for example, the number and size of the icecrystals strongly influences the drying properties of the lyophilizedcake. Larger ice crystals produced by warmer nucleation temperaturesleave behind larger pores upon sublimation, and the larger pores offerless resistance to the flow of water vapor during subsequentsublimation. Hence, the present methods provide a means of increasingprimary drying (i.e., sublimation) rates in freeze-drying processes byincreasing the nucleation temperature.

Another possible benefit may be realized in applications where sensitivematerials are preserved via freezing processes (i.e., cryopreserved).For example, a biological material including but not limited to,mammalian tissue samples (e.g., cord blood, tissue biopsy, egg and spermcells, etc.), cell lines (e.g., mammalian, yeast, prokaryotic, fungal,etc.) and biological molecules (e.g., proteins, DNA, RNA and subclassesthereof) frozen in an aqueous solution may experience various stressesduring the freezing process that may impair the function or activity ofthe material. Ice formation may physically disrupt the material orcreate severe changes in the interfacial bonding, osmotic forces, soluteconcentrations, etc. experienced by the material. Since nucleationcontrols the structure and kinetics of ice formation, it cansignificantly influence these stresses. The presently disclosed methodstherefore provides a unique means of mitigating stresses associated withcryopreservation processes and enhancing the recovery of function oractivity from cryopreserved materials. The present methods alsorepresent improvement over conventional nucleation control methods(e.g., seeding or contact with cold surfaces) used to initiateextracellular ice formation in two-step cryopreservation algorithmsdesigned for living cells for small to large commercial scale.

As an example of this improved biological activity resulting from thepresent process, Jurkat A3 lymphoblasts (ATCC No. CRL-2570) obtainedfrom ATCC (Manassas, Va.) were subjected to the present process. Stockcultures of the Jurkat A3 lymphoblasts were maintained at 37° C. in 95%air and 5% CO₂ in Falcon T-75 cm² flasks using a complete RPMI-1640media containing 2.5 mM glutamine (HyClone) and supplemented with 10%v/v FetalClone III serum (HyClone). For cell expansion, stock cultureswere sub-cultured every 2 days when cell density reached approximately5×10⁶ cells/ml and then re-suspended in fresh T-75 flasks at a seedingdensity of about 0.5×10⁶ cells/ml. For the Jurkat cell line, an in-housemedia/DMSO solution was prepared by the addition of 5% v/v DMSO to thecomplete RPMI-1640 media containing 20% v/v FetalClone III.

Briefly, the Jurkat A3 lymphoblast cells were processed after cellexpansion for preservation in bags by pooling cultures and re-suspendingthem to a cell density of 2×10⁶ cells/ml. Cryopreservation studiesemploying present process were performed using the advanced controlledrate freezing system designed for bags and used to process the Jurkat A3lymphoblast cell line. Samples were held for 10 min at 2-8° C. in theadvanced controlled rate freezing chamber prior to freezing to allowcell samples to equilibrate. Following the freezing program, sampleswere immediately transferred to liquid nitrogen for up to 48 hrs.Samples were thawed in a 37° C. water bath, immediately re-suspended inappropriate culture media (1:10 dilution), plated, and allowed torecover prior to cell viability assessment.

Cell viability of the cryopreserved Jurkat A3 lymphoblast cells wasassessed both qualitatively and quantitatively prior to freezing andpost-thaw. For both cell lines, qualitative assessment was achieved byvisualization using light microscopy. For the Jurkat A3 lymphoblast cellline, quantitative assessment was accomplished using Trypan Bluestaining through an automated CEDEX cell analyzer (Roche AppliedScience). After thawing, a sample from the 25 ml bag was re-suspended infresh media and allowed to recover in well plates prior to viabilityassessments at 3 hrs and 24 hrs post-preservation. From each well, asample was taken and directly measured using the CEDEX cell counter.

As discussed above, the Jurkat A3 lymphoblast cell recovery was assessedat 3 hr and at 24 hr post-thaw viability following freezing in theadvanced controlled rate freezing system with and without nucleationcontrol, see FIG. 32. For the evaluated trials, the bags containing saidmaterials containing the live cells were equilibrated at 4° C. in theadvanced controlled rate freezing process within the controlled ratefreezing chamber prior to freezing. For the case without nucleationcontrol, after equilibration the bags were cooled to −45° C. at acooling rate of 1° C./min and then further cooled to −100° C. at acooling rate of 10° C./min For the case of cold spike nucleationcontrol, after equilibration the bags went through the following steps:(i) cooled to −5° C. at a cooling rate of 1° C./min, (ii) nucleated by acold spike, (iii) warmed to −20° C. at a warming rate of 10° C./min,(iv) cooled to −45° C. at a cooling rate of 1° C./min, and (v) cooled to−100° C. at a cooling rate of 10° C./min. In FIG. 32, the results showthat the cryopreservation algorithm with controlled nucleation yieldedapproximately 15% more cell recovery at about 3 hrs post-thaw andapproximately 10% more cell recovery at 24 hrs post-thaw when comparedto the cryopreservation algorithm with uncontrolled nucleation. Thiscryopreservation example shows the significant impact of nucleationcontrol behavior and thus the freezing process uniformity on cellrecovery post-thaw.

The presently disclosed controlled rate freezing and nucleation controlmethods may be also applied to complex solutions or mixtures containingseveral constituents both in cryopresevation and lyophilizationapplications. These formulations are often solutions with an aqueous,organic, or mixed aqueous-organic solvent containing a pharmaceuticallyactive ingredient (e.g., a synthetic chemical, protein, peptide, orvaccine) and optionally, one or more mitigating constituents, includingbulking agents that help prevent physical loss of the active ingredientduring drying (e.g., dextrose, glucose, glycine, lactose, maltose,mannitol, polyvinyl pyrrolidone, sodium chloride, and sorbitol);buffering agents or toxicity modifiers that help maintain theappropriate environmental pH or toxicity for the active constituent(e.g., acetic acid, benzoic acid, citric acid, hydrochloric acid, lacticacid, maleic acid, phosphoric acid, tartaric acid, and the sodium saltsof the aforementioned acids); stabilizing agents that help preserve thestructure and function of the active constituent during processing or inits final liquid or dried form (e.g., alanine, dimethylsulfoxide,glycerol, glycine, human serum albumin, polyethylene glycol, lysine,polysorbate, sorbitol, sucrose, and trehalose); agents that modify theglass transition behavior of the formulation (e.g., polyethylene glycoland sugars), and anti-oxidants that protect the active constituent fromdegradation (e.g., ascorbate, sodium bisulfite, sodium formaldehyde,sodium metabisulfite, sodium sulfite, sulfoxylate, and thioglycerol).

Since nucleation is typically a random process, a plurality of the samematerial subjected to identical processing conditions might nucleate atdifferent temperatures. As a result, the properties of those materialsthat depend on nucleation behavior will likely differ despite theidentical processing conditions. The disclosed methods provide a meansfor controlling the nucleation temperatures of a plurality of materialssimultaneously and thereby offers a way to increase the uniformity ofthose product properties that depend on nucleation behavior. In atypical freeze-drying process, for example, the same solution inseparate vials may nucleate stochastically over a wide range oftemperatures, and as a result, the final freeze-dried products maypossess significant variability in critical properties like residualmoisture, activity and reconstitution time. By controlling thenucleation temperature via the presently disclosed process, thevial-to-vial uniformity of product properties from a freeze-drying canprocess can be dramatically improved.

The ability to control the nucleation behavior of a material may alsoprovide substantial benefit in reducing the time necessary to develop anindustrial process that hinges upon a normally uncontrolled nucleationevent. For example, it often takes many months to develop a successfulfreeze-drying cycle that can be accomplished in a reasonable amount oftime, yields desired product properties within the specified uniformity,and preserves sufficient activity of the active pharmaceuticalingredient (API). By providing a means of controlling nucleation andthereby potentially improving primary drying time, product uniformity,and API activity, the present methods should dramatically reduce thetime necessary to develop successful freeze-drying protocols.

In particular, the potential benefits of the present nucleation processprovide increased flexibility in specifying the composition of theformulation to be freeze-dried. Since controlled nucleation can betterpreserve the API during the freezing step, users should be able tominimize the addition of mitigating constituents (e.g., stabilizingagents) to the formulation or chose simpler combinations of formulationconstituents to achieve combined stability and processing goals.Synergistic benefits may arise in cases where controlled nucleationminimizes the use of stabilizing agents or other mitigating constituentsthat inherently lengthen primary drying times (e.g., by decreasing glasstransition temperatures of aqueous solutions).

The disclosed methods are particularly well-suited for large scaleproduction or manufacturing operations since it can be conducted usingthe same equipment and process parameters that can easily be scaled oradapted to manufacture a wide range of products. The process providesfor the nucleation of materials using a process where all manipulationscan be carried out in a single chamber (e.g., a freeze-dryer) and wherethe process does not require use of a vacuum, use of additives,vibration, electrofreezing or the like to induce nucleation.

In contrast to the prior art, the present method does not add anythingto the lyophilized product. It only requires that the materials, (e.g.,liquids in the vials), be held initially at a specified pressure under agas environment and that the pressure is rapidly reduced to a lowerpressure or the temperature is uniformly controlled to inducenucleation. Any applied gas will be removed from the vials during thelyophilization cycle. The vials or their contents are not contacted ortouched with anything except the gas. Simple manipulation of the ambientpressure and gas environment or the uniform cryogenic cold gastemperature is sufficient on its own to achieve that goal. By relyingonly on ambient pressure change to induce nucleation or a temperaturechange of the uniform cryogenic cold gas, the present method disclosedherein uniformly and simultaneously affects all vials within afreeze-dryer or uniform flow controlled rate freezer.

The present embodiment is also less expensive and easier to implementand maintain than prior art methods of influencing nucleation inmaterials in lyophilization applications. The present method enablessignificantly faster primary drying in lyophilization processes, therebyreducing processing costs for freeze-dried pharmaceuticals. The presentmethod produces much more uniform lyophilized products than prior artmethods, thereby reducing product losses and creating barriers to entryfor processors unable to meet tighter uniformity specifications. Thismethod achieves these benefits without contaminating the lyophilizedproduct. Greater process control should lead to an improved product andshortened process times.

While the present teachings have been described in conjunction withvarious embodiments and examples, it is not intended that the presentteachings be limited to such embodiments or examples. On the contrary,the present teachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.Accordingly, the foregoing description and drawings are by way ofexample only.

1. A method of controlling a chilling or freezing process of biologicalmaterial disposed in a plurality of containers, comprising the steps of:(i) placing said plurality of containers of said biological materials ina cooling area defined as an area between a gas distribution surface anda parallel gas collection surface within a cooling chamber; (ii) mixinga liquid cryogen with a warmer gas to produce a cold cryogenic gas at aselected temperature profile, said temperature profile corresponding toa desired cooling rate of said biological materials within saidcontainers; (iii) delivering a unidirectional flow of said coldcryogenic gas through the gas distribution surface to said cooling areabetween said parallel gas distribution and said gas collection surfacesand generally parallel to each of said plurality of containers touniformly cool said biological materials within said containers; and(iv) promptly exhausting said gas from said cooling chamber via said gascollection surface so as to prevent recirculation of said gas withinsaid cooling area; wherein greater uniformity of said biologicalmaterials in each of said plurality of containers is achieved.
 2. Themethod of claim 1, wherein said collection surface and/or said gasdistribution surface are porous surfaces.
 3. The method of claim 1,wherein said step of mixing liquid cryogen with said warmer gas furthercomprises mixing liquid nitrogen with either room temperature nitrogengas from a nitrogen supply source or with recycled gas exiting from thecooling chamber or a combination thereof and said mixing occurs incryogen intake circuits to produce a cold nitrogen gas at said selectedtemperature profile, said temperature profile corresponding to a desiredcooling rate of said biological materials within said containers.
 4. Themethod of claim 1 wherein the step of delivering a unidirectional flowof said cold cryogenic gas further comprises performing a temperaturequench of said biological material in each of said plurality ofcontainers by delivering a unidirectional flow of cold cryogenic gashaving a temperature of 40° C. or more below the temperature of saidbiological material in said plurality of containers to induce nucleationof freezing in said biological materials.
 5. The method of claim 1further comprising the step of rapidly reducing the pressure in thecooling chamber during the step of delivering a unidirectional flow ofsaid cold cryogenic gas to induce nucleation of freezing in saidbiological materials.
 6. The method of claim 1 wherein greateruniformity of said biological materials further comprises greateruniformity of the cell viability of said biological materials in saidplurality of containers.
 7. The method of claim 1 wherein greateruniformity of said biological materials further comprises greateruniformity of the biological activity of said biological materials insaid plurality of containers.
 8. The method of claim 1, wherein saiddesired cooling rate of said biological materials within said containersis between about −2.5° C./min to about −5.0° C./min.
 9. The method ofclaim 1, wherein said plurality of containers comprise at least 10,000vials.
 10. The method of claim 1, wherein said plurality of containerscomprise at least 50,000 vials.
 11. The method of claim 1, wherein saidplurality of containers comprise a plurality of bags.
 12. The method ofclaim 1, wherein said biological material in each of a plurality ofcontainers comprises; microorganisms, tissues, organs, stem cells,primary cells, cell lines, small multicellular organisms, complexcellular structures, live or attenuated viruses, nucleic acids,monoclonal antibodies, polyclonal antibodies, biomolecules, non-peptideanalogues, peptides, proteins, RNA, DNA, oligonucleotides, and/or viralparticles.
 13. A cooling unit, comprising a uniform flow cryogenicchiller including a cryogen intake circuit coupled to a source ofcryogen wherein said cryogenic chiller further includes a base gasinjection box, a porous metal plate disposed or set in or near the topsurface of said gas injection box, and a corresponding gas removal boxpositioned immediately above said base gas injection box with saidporous metal plate disposed therein.
 14. A cooling unit comprising achilling or freezing control system for controlling a cryogen source, anintake circuit coupled to said cryogen source and adapted for providinga uniform flow and temperature of a cryogenic cold gas to said coolingchamber, and wherein said cooling unit also comprises an intake plenum,an exhaust manifold, and two or more parallel porous surfaces thatdefine a cooling area between adjacent parallel surfaces with one ofsaid parallel porous surfaces disposed adjacent to said intake plenumand in fluid communication with said intake plenum and another of saidparallel porous surfaces disposed adjacent to said exhaust manifold,said parallel porous surfaces and associated cooling area adapted toretain, or hold, a plurality of containers of biological materials.